Compounds and methods of treating bacterial infections

ABSTRACT

The invention relates to compounds of Formula (I), wherein the variables are as defined in the claims, which are useful in the treatment and/or prevention of bacterial infections in a subject. The invention further relates to the use of a compound of Formula (I) in the manufacture of a medicament, and medical devices when used in a method of treating or preventing a bacterial infection in a subject, and related aspects.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. 371 National Phase Entry Applicationfrom PCT/SE2016/051207, filed Dec. 2, 2016, which claims the benefit ofSwedish Patent Application No. 1551572-9 filed on Dec. 2, 2015, thedisclosure of which is incorporated herein in its entirety by reference.

TECHNICAL FIELD

This invention relates to compounds of Formula I, methods of treating,eliminating and/or preventing a bacterial infection in a subject using acompound of Formula I, the use of a compound of Formula I in themanufacture of a medicament for the treatment of a bacterial infectionin a subject, and medical devices when used in a method of treating orpreventing a bacterial infection in a subject.

BACKGROUND ART

A marked increase in prevalence of multi-drug resistance indisease-causing Gram-positive G+ve (inter alia Staphylococcus aureus,Enterococcus spp., Streptococcus pneumoniae, Clostridium difficile,Mycobacterium tuberculosis) and Gram-negative bacteria (inter aliaKlebsiella spp., Neisseria gonorrhoeae, Acinetobacter spp.,Campylobacter spp., Enterobacter spp., Pseudomonas aeruginosa,Salmonella spp., Shigella spp.) has coincided with an unprecedentedglobal decline in investment in new anti-infective drugs. There are fewcurrently registered alternatives for multidrug resistant (MDR)bacterial infections, forcing clinicians to consider older generationdrugs such as colistin with narrow spectrum and considerable potentialfor toxic side-effects. In addition, there are fewer novel classes ofanti-infective therapeutics moving through the drug developmentpipeline.

Since the year 2000, a period of almost 15 years, only 5 novel mode ofaction (MOA) antibacterial agents have been approved by the USFDA—linezolid (an oxazolidinone) in 2000, daptomycin (a lipopeptide) in2003, retapamulin (a pleuromutilin) in 2007, fidaxomicin (a macrolidetiacumicin) in 2011, and bedaquiline (a diarylquinoline) in 2012. Nonovel MOA antibacterial agents were approved in 2013 and to date in 2014only tedizolid and dalbavancin, both analogues of existing classes, havebeen recommended for approval in the US. While there are more than 300anti-infective medicines in various stages of development, the largemajority of these medicines are previously approved antibacterialcompounds or their derivatives that are undergoing studies for newindications.

Furthermore, the prevalence of multidrug-resistance in animal-specificpathogens together with greater regulation of the registration and usageof antimicrobials in animals, has caused veterinarians to becomeincreasingly reliant on the traditional classes of antimicrobial agents.The risk of transfer of MDR zoonotic organisms from animals to humanshas also led to calls for further restrictions on the usage of somerecently registered antibacterial drugs such as the fluoroquinolones andthe third and fourth generation cephalosporins.

Epidemiology of Antibacterial Resistance Development in Pathogens ofHumans and Animals

Much of the evolution in resistance development is driven by changes inthe epidemiology of key MDR organisms. Once only restricted to humanhospitals and aged care facilities, methicillin resistant Staphylococcusaureus (MRSA) strains are now being isolated from the community inalarming proportions. Furthermore, community-acquired MRSA strains aremore likely to carry the Panton-Valentine leukocidin (PVL) toxin, avirulence factor linked to skin and soft tissue lesions as well as arapid, fulminating, necrotizing pneumonia with significant associatedmortality. Recently MRSA strains have become host-adapted in several keyanimal species including livestock, horses and companion animals andregular cases of human-to-animal and animal-to-human transfer are beingdocumented. This has important consequences for strain transmission andpublic health. A recent survey of 751 Australian veterinarians for MRSAnasal carriage found that a remarkable 21.4% of equine veterinarianswere MRSA-positive compared to 4.9% of small animal veterinarians and0.9% of veterinarians with little animal contact. These ecologicalshifts of MRSA together with the emergence of resistance to new drugsdeveloped specifically for MRSA such as linezolid, confirm that new MRSAanti-infectives are urgently needed. Furthermore, hospitals that usevancomycin for treating MRSA then have to contend with outbreaks ofvancomycin-resistant enterococci (VRE) infections in their patients,once again with limited alternative antimicrobial choices.

The World Health Organisation has identified antibiotic resistance asone of the three major future threats to global health. A recent reportfrom the US Centers for Disease Control and Prevention (CDC) estimatedthat “in the United States, more than two million people are sickenedevery year with antibiotic-resistant infections, with at least 23,000dying as a result.” The extra medical costs, in the USA alone,associated with treating and managing a single case ofantibiotic-resistant infection are estimated to be between US$18,588 andUS$29,069 per year resulting in an overall direct cost to the US healthsystem of over US$20 billion annually. In addition, the cost to UShouseholds in terms of lost productivity is estimated at over US$35billion per annum. Twenty five thousand patients in the European Union(EU) still die annually from infection with MDR bacteria despite many EUcountries having world's best practice hospital surveillance andinfection control strategies. The EU costs from health care expenses andlost productivity associated with MDR infections are estimated to be atleast €1.5 billion per year.

There is an unmet clinical need for antibacterial agents with novelmechanisms of action to supplement and replace currently availableantibacterial agents, the efficacy of which is increasingly underminedby antibacterial resistance mechanisms. There additionally remains aneed for alternative antibacterials in the treatment of infection bymulti-resistant bacteria. However, as reported by the InfectiousDiseases Society of America and the European Centre for Disease Controland Prevention, few new drugs are being developed that offer promisingresults over existing treatments (Infectious Diseases Society of America2010, Clinical Infectious Diseases, 50(8): 1081-1083).

Ayer W A & Villar J D F Can J Chem 63, 1161 (1985) describe an enolicnatural product isolated from the fungus Lachnellula fuscosanguinea,which they term lachnellulic acid:

The compound is active against the Dutch Elm disease fungus. Note thatthe alkyl chain adjacent the enolic keto function is longer than thechain adjacent the lactone.

Kong et al, J. Nat. Prod. 2005 68, 920-923 describe natural productsisolated from a Pseudomonas species associated with a Pacific marinesponge, which they term pseudopyronines A & B:

The compounds are described as having “moderate to poor” antibacterialactivities, but are unstable and degrade to a hydroxyfuranone species.Note that the pyranone ring has two unsaturated bonds. In a relatedpaper, Singh et al J Antibiotics 56 No 12 1033-1044 (2003), theantibacterial activity of the compounds is believed to derive frominhibition of the bacterial fatty acid biosynthesis pathway, in asomewhat similar fashion to the antibacterial agent triclosan.

Miyakado et al Chem Lett 10 1539-42 (1982) describes the isolation ofnatural products from the plant Podophyllum peltatum, which they termpodoblastin A:

The compounds are said to be active against the fungus Pyriculariaoryziae. Note that the alkyl chain adjacent the carbonyl is markedlylonger than the alkyl chain adjacent the lactone.

The discussion of the background art set out above is intended tofacilitate an understanding of the present invention only. Thediscussion is not an acknowledgement or admission that any of thematerial referred to is or was part of the common general knowledge asat the priority date of the application.

SUMMARY OF INVENTION

According to one aspect of the invention, there is provided a compoundof Formula I, or a stereoisomer, tautomer, pharmaceutically acceptablesalt, or prodrug thereof:

whereinR¹ is C₁-C₂₀alkyl, C₂-C₂₀alkenyl, C₂-C₂₀alkynyl, C₁-C₆alkoxyC₁-C₃alkylor C₃-C₆ cycloalkylC₁-C₃alkyl, in which any alkyl, alkenyl, alkynyl orcycloalkyl is optionally substituted with up to three substituentsindependently selected from halo, —OR₅, —C(O)OR⁵, —C(O)R⁵, —OC(O)OR⁵,—NR⁵R⁶, —C(O)NR⁵R⁶, —OC(O)NR⁵R⁶, nitro, cyano and azido, and in the caseof cycloalkyl, also C₁-C₆alkyl or C₁-C₆haloalkyl;R² is C₁-C₂₀alkyl, C₂-C₂₀alkenyl, C₂-C₂₀alkynyl, C₁-C₆alkoxyC₁-C₃alkyl,C₁-C₆ cycloalkylC₁-C₃alkyl or C₁-C₆cyloalkenylC₁-C₃alkyl, in which anyalkyl, alkenyl, alkynyl, cycloalkyl or cycloalkenyl is optionallysubstituted with up to three substituents independently selected fromhalo, —OR⁵, —C(O)OR⁵, —C(O)R⁵, —OC(O)OR⁵, —NR⁵R⁶, —C(O)NR⁵R⁶,—OC(O)NR⁵R⁶, nitro, cyano and azido, and in the case of cycloalkyl, alsoC₁-C₆alkyl or C₁-C₆haloalkyl;R³ is a bond along with the adjacent carbon defining a keto group, R⁵,C(O)R⁵, CR^(a)R^(b)OR⁵, CR^(a)R^(b)OC(O)R⁵, CR^(a)R^(b)OC(O)OR⁵ orC(O)CHR⁷NH₂;R⁴ is R⁵, C(O)R⁵, CR^(a)R^(b)OR⁵, CR^(a)R^(b)OC(O)R⁵,CR^(a)R^(b)OC(O)OR⁵ or C(O)CHR⁷RNH₂;R⁵ and R⁶ are independently selected from H, C₁-C₆alkyl, C₃-C₆cycloalkyland C₃-C₆cycloalkylC₁-C₃alkyl, wherein the C₁-C₆alkyl or cycloalkyl isoptionally substituted with halo, hydroxy, C₁-C₄alkoxy, C₁-C₄haloalkyl,hydroxyC₁-C₄alkyl, C₁-C₄alkylcarbonyl, SH, SMe, COOH, COOC₁-C₄alkyl andCONH₂;R⁷ is H, C₁-C₆alkyl, C₃-C₆cycloalkyl and C₃-C₆cycloalkylC₁-C₃alkyl,wherein the C₁-C₆alkyl or C₃-C₆cycloalkyl is optionally substituted withhalo, hydroxy, phenyl, C₁-C₄alkoxy, C₁-C₄haloalkyl, hydroxyC₁-C₄alkyl,C₁-C₄alkylcarbonyl, SH, SMe, COOH, COOC₁-C₄alkyl and CONH₂;R^(a) and R^(b) are independently selected from H and methyl;or a pharmaceutically acceptable salt, stereoisomer, N-oxide or hydratethereof; with the proviso that when R³ is a bond, R⁴ is H, and R¹ and R²are both unsubstituted alkyl, then if R¹ is C₆-alkyl, R² has at leasttwo carbon atoms.

In certain embodiments, R¹ is C₁-C₂₀alkyl which is optionallysubstituted as defined above. In sub-embodiments, R¹ is C₆-C₁₂alkyl,including n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl and n-dodecyl,especially n-nonyl, any of which alkyl species being optionallysubstituted with 1, 2 or 3 substituents independently selected fromC₁-C₄alkyl, C₁-C₄haloalkyl, halo, hydroxy, C₁-C₄alkoxy,C₁-C₄hydroxyalkyl and C₁-C₄alkylcarbonyl.

Further, R¹ may be selected from the group consisting of C₂-C₂₀alkyl;C₃-C₂₀alkyl; C₄-C₂₀alkyl; C₅-C₂₀alkyl; C₆-C₂₀alkyl; C₇-C₂₀alkyl; andC₈-C₂₀alkyl. In this embodiment, R² may be selected from the groupconsisting of C₂-C₁₉alkyl; C₂-C₁₈alkyl; C₂-C₁₇alkyl; C₂-C₁₆alkyl;C₂-C₁₅alkyl; C₂-C₁₄alkyl; and C₂-C₁₃alkyl. Thus, advantageously, ashorter R¹ substituent may be combined in Formula I with a longer R²substituent, whether it is alkyl, alkenyl or alkynyl. The herein definedranges for R¹ and R² may be combined with anyone of the herein definedother R substituents.

In certain embodiments R¹ is C₂-C₂₀alkenyl comprising one to threeunsaturated bonds which is optionally substituted as defined above. Insub-embodiments, R¹ is C₆-C₁₂alkenyl, such as hexenyl, heptenyl,octenyl, decenyl, undecenyl or dodecenyl, including those with oneunsaturated bond, and especially those in the 1-position.

The bond connecting R¹ to the pyran ring is chiral, and the inventionincludes certain embodiments which are racemic at this position, orembodiments enantiomerically enriched with the S or R diastereomer, forexample 75% enantiomerically enriched as regards one diastereomer,typically greater than 90% enantiomerically enriched, such as greaterthan 95% enantiomerically enriched.

In certain embodiments, R² is C₁-C₂₀alkyl which is optionallysubstituted as defined above. In sub-embodiments, R¹ is C₁-C₆alkyl,including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl and n-hexyl, any of which alkyl speciesbeing optionally substituted with 1, 2 or where valance permits 3substituents independently selected from C₁-C₄ alkyl, C₁-C₄ haloalkyl,halo, hydroxy, C₁-C₄alkoxy, C₁-C₄hydroxyalkyl and C₁-C₄alkylcarbonyl.

In certain embodiments, R² is C₁-C₆cycloalkylC₁-C₃alkyl which isoptionally substituted as defined above. Subembodiments includecyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl and especiallycycloehexylmethyl, any of which cycloalkyl species being optionallysubstituted with 1, 2 or where valance permits 3 substituentsindependently selected from C₁-C₄alkyl, C₁-C₄haloalkyl, halo, hydroxy,C₁-C₄alkoxy, C₁-C₄hydroxyalkyl and C₁-C₄alkylcarbonyl.

An additional proviso may be applied to Formula I, wherein when R³ is abond, R⁴ is H, and R¹ and R² are both unsubstituted C₁-C₂₀ alkyl, thenR¹ has at least 2 more carbon atoms than R².

In an advantageous embodiment, R² is a C₃alkyl, combined with any one ofthe herein discussed other substituents following the definedproviso(s).

The compound according to the invention may be selected from the groupconsisting of:

-   3-butyryl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   (R)-3-butyryl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   (S)-3-butyryl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   6-decyl-4-hydroxy-3-pentanoyl-5,6-dihydro-2H-pyran-2-one,-   (E)-3-butyryl-6-(non-1-en-1-yl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-6-nonyl-3-propionyl-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-6-octyl-3-propionyl-5,6-dihydro-2H-pyran-2-one,-   3-acetyl-4-hydroxy-6-octyl-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-6-nonyl-3-pentanoyl-5,6-dihydro-2H-pyran-2-one,-   3-acetyl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   6-decyl-4-hydroxy-3-propionyl-5,6-dihydro-2H-pyran-2-one,-   3-acetyl-6-decyl-4-hydroxy-5,6-dihydro-2H-pyran-2-one,-   3-butyryl-6-decyl-4-hydroxy-5,6-dihydro-2H-pyran-2-one,-   3-butyryl-4-hydroxy-6-octyl-5,6-dihydro-2H-pyran-2-one,-   3-(2-cyclohexylacetyl)-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-3-(3-methylbutanoyl)-6-nonyl-5,6-dihydro-2H-pyran-2-one-   3-butyryl-4-hydroxy-6-undecyl-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-3-pentanoyl-6-undecyl-5,6-dihydro-2H-pyran-2-one,-   3-hexanoyl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   8-(5-butyryl-4-hydroxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)octanenitrile,-   3-butyryl-4-hydroxy-6-(7-methoxyheptyl)-5,6-dihydro-2H-pyran-2-one,-   3-butyryl-6-(2,6-dimethylhept-5-enyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one,-   3-butyryl-6-(cyclopentylmethyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one,-   3-(2-cyclopentylacetyl)-4-hydroxy-6-(6-methoxyhexyl)-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-3-(3-methoxypropanoyl)-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   3-(2-cyclopropylacetyl)-4-hydroxy-6-(6-methoxyhexyl)-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-6-nonyl-3-(2-(tetrahydro-2H-pyran-4-yl)acetyl)-5,6-dihydro-2H-pyran-2-one,-   8-(4-hydroxy-6-oxo-5-(2-(tetrahydro-2H-pyran-4-yl)acetyl)-3,6-dihydro-2H-pyran-2-yl)octanenitrile,    and-   8-(5-(2-cyclohexylacetyl)-4-hydroxy-6-oxo-3,6-dihydro-2H-pyran-2-yl)octanenitrile,    or a pharmaceutically acceptable salt, stereoisomer, N-oxide or    hydrate thereof.

In one embodiment, the compound is selected from the group consistingof:

-   3-butyryl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   (R)-3-butyryl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   (S)-3-butyryl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   6-decyl-4-hydroxy-3-pentanoyl-5,6-dihydro-2H-pyran-2-one,-   (E)-3-butyryl-6-(non-1-en-1-yl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-6-nonyl-3-propionyl-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-6-octyl-3-propionyl-5,6-dihydro-2H-pyran-2-one,-   3-acetyl-4-hydroxy-6-octyl-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-6-nonyl-3-pentanoyl-5,6-dihydro-2H-pyran-2-one,-   3-acetyl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   6-decyl-4-hydroxy-3-propionyl-5,6-dihydro-2H-pyran-2-one,-   3-acetyl-6-decyl-4-hydroxy-5,6-dihydro-2H-pyran-2-one,-   3-butyryl-6-decyl-4-hydroxy-5,6-dihydro-2H-pyran-2-one,-   3-butyryl-4-hydroxy-6-octyl-5,6-dihydro-2H-pyran-2-one,-   3-(2-cyclohexylacetyl)-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-3-(3-methylbutanoyl)-6-nonyl-5,6-dihydro-2H-pyran-2-one-   3-butyryl-4-hydroxy-6-undecyl-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-3-pentanoyl-6-undecyl-5,6-dihydro-2H-pyran-2-one,-   3-hexanoyl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   3-butyryl-6-(2,6-dimethylhept-5-enyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one,-   3-butyryl-6-(cyclopentylmethyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-3-(3-methoxypropanoyl)-6-nonyl-5,6-dihydro-2H-pyran-2-one,-   4-hydroxy-6-nonyl-3-(2-(tetrahydro-2H-pyran-4-yl)acetyl)-5,6-dihydro-2H-pyran-2-one,    and-   8-(4-hydroxy-6-oxo-5-(2-(tetrahydro-2H-pyran-4-yl)acetyl)-3,6-dihydro-2H-pyran-2-yl)octanenitrile;    or a pharmaceutically acceptable salt, stereoisomer, N-oxide or    hydrate thereof.

The present inventors have found that advantageous properties may befound with compounds defined by Formula I which comprise at least onerelatively long carbon chain in either the R¹ or the R² position. Thus,for example, if R¹ is C₇-C₂₀alkyl, then R² may be shorter such asC₂alkyl or C₃alkyl. Alternatively, if R² is is C₇-C₂₀alkyl, then R¹ maybe shorter such as C₂alkyl or C₃alkyl. When the length of R1 is heldconstant in a series of compounds, the activity of the compounds appearsto increase with the length of R2, and vice versa.

R³ may be a bond to the adjacent carbon thereby defining a keto group.If R³ defines such a keto group, then R⁴ may advantageously be H, andthe above-discussed relationships between the chain length of R¹ and R²may apply.

In other embodiments R³ is H, it being appreciated that when R³ is H, adepiction of the compound as a delocalized structure as illustratedbelow is appropriate:

Embodiments in which R³ is H in the therapeutic form represent aconvenient handle to allow the compound to be derivatised withconventional prodrug moieties, such as C(O)R⁵ (for example where R⁵ ismethyl, isopropyl, cyclopropyl or tert-butyl, any of which may beoptionally fluorinated), —CR^(a)R^(b)OR⁵, (for example where R^(a) andR^(b) are both H or both methyl and R⁵ is methyl, isopropyl, cyclopropylor tert-butyl, any of which may be fluorinated), —CR^(a)R^(b)OC(O)R⁵,(for example where R^(a) and R^(b) are both H or both methyl and R⁵ ismethyl, isopropyl, cyclopropyl or tert-butyl, any of which may beoptionally fluorinated), —CR^(a)R^(b)OC(O)OR⁵, (for example where R^(a)and R^(b) are both H or both methyl and R⁵ is methyl, isopropyl,cyclopropyl or tert-butyl, any of which may be optionally fluorinated)or —C(O)CHR⁷NH₂, wherein R⁷ is as stated above, for example R⁷ is theside chain of an L-amino acid such as the side chain of alanine, valine,leucine or isoleucine. In alternative embodiments R³ remains bound tothe compound of formula I in its therapeutic form, for example when R³is C₁-C₆alkyl, (for example methyl, ethyl, isopropyl, cyclopropyl ortert-butyl, any of which may be optionally fluorinated)

R⁴ may be H. Compounds wherein R⁴ is H in the therapeutic form representa convenient handle to allow the compound to be derivatised withconventional prodrug moieties, such as C(O)R⁵ (for example where R⁵ ismethyl, isopropyl, cyclopropyl or tert-butyl, any of which may beoptionally fluorinated), —CR^(a)R^(b)OR⁵, (for example where R^(a) andR^(b) are both H or both methyl and R⁵ is methyl, isopropyl, cyclopropylor tert-butyl, any of which may be fluorinated), —CR^(a)R^(b)OC(O)R⁵,(for example where R^(a) and R^(b) are both H or both methyl and R⁵ ismethyl, isopropyl, cyclopropyl or tert-butyl, any of which may beoptionally fluorinated), —CR^(a)R^(b)OC(O)OR⁵, (for example where R^(a)and R^(b) are both H or both methyl and R⁵ is methyl, isopropyl,cyclopropyl or tert-butyl, any of which may be optionally fluorinated)or —C(O)CHR⁷NH₂, wherein R⁷ is as stated above, for example R⁷ is theside chain of an L-amino acid such as the side chain of alanine, valine,leucine or isoleucine. In alternative embodiments R³ remains bound tothe compound of formula I in its therapeutic form, for example when R³is C₁-C₆alkyl, (for example methyl, ethyl, isopropyl, cyclopropyl ortert-butyl, any of which may be optionally fluorinated).

Alternatively, R⁴ may be e.g. a methyl. If R⁴ is a methyl, then at leastone of R¹ and R² is longer than 3 carbons.

According to another aspect of the invention, there is provided a methodof treating, eliminating and/or preventing a bacterial colonisation orinfection in a subject, the method comprising the step of administeringa therapeutically effective amount of a compound of Formula I, or atherapeutically acceptable salt thereof, to the subject. The method oftreating, eliminating and/or preventing a bacterial infection orcolonisation in a subject, may also comprise the administration of thepharmaceutical or veterinary compositions of the invention. Of especialinterest is the decontamination of patients carrying MRSA by topical useof a compound of Formula I.

In the present application, the term “colonization” is understood tomean bacterial growth on any surface, such as a skin or other surface ofa subject, such as a human, or the surface of implants or other medicalequipment. Bacterial colonization may also be prevented using thepresent invention on any other objects which are at risk of obtainingsuch bacterial growth, such as other hospital equipment, furniture,clothing etc.

The pharmaceutical composition comprising a compound according to theinvention may e.g. be used for decolonisation of skin as a preoperativemeasure for surgical site infection prevention.

The invention also embraces any one of the compounds of Formula I foruse as a medicament.

Thus, the invention relates to e.g. a compound defined by Formula I,wherein R³ is a bond, R⁴ is H, R¹ is C₆-alkyl and R² is a methyl for useas a medicament.

According to a further aspect of the invention, there is provided theuse of a compound of Formula I, or a therapeutically acceptable saltthereof, in the manufacture of medicament the treatment of a bacterialcolonisation or infection in a subject.

The subject may be any subject capable of colonisation and infection bybacteria. The subject may be mammalian, or may be piscine or avian.Preferably, the subject is selected from the group comprising, but notlimited to, human, canine, feline, bovine, ovine, caprine, otherruminant species, porcine, equine, avian, or piscine.

The compound of Formula I may be administered to the subject in a doseselected from the group comprising 0.1 mg/kg to 250 mg/kg body weight,typically 1 mg/kg to 100 mg/kg body weight, and more generally 5 mg/kgto 50 mg/kg body weight. Titration of dosage range to suit the infectionand size and physical condition of the patient is readily achieved bythe skilled general practitioner or veterinary physician. The compoundof Formula I may be administered to the subject using a dosing scheduleselected from the group consisting of: hourly, 3 times daily; twicedaily; daily; every second day; twice weekly; once weekly; oncefortnightly; once monthly; once every two months or by constant rate orvariable rate infusion. Preferably, the compound of Formula I isadministered until colonisation or the signs and symptoms of infectionor colonisation have at least been partially treated or alleviated.

In one embodiment, the concentration of compound of Formula I (or ametabolite) in the subject's blood after treatment is within a rangeselected from the group comprising, but not limited to: between 0.1 and10 μg/mL at 2 hours, 1 and 200 μg/mL after 12 hours; between 0.1 and 5μg/mL after 24 h; between 0.01 and 2 μg/mL after 48 hours; between0.0001 and 1 μg/mL after 72 h. Preferably, the concentration is selectedfrom the group comprising, but not limited to: less than 200 μg/mL after12 hours; less than 5 μg/mL after 24 hours; less than 1 μg/L after 48hours and less than 0.5 μg/mL after 72 hours. Selection of therapeuticdosing range and regime and titration to suit the particular infectionand size and physical condition of the patient is readily achieved bythe skilled general practitioner or veterinary physician.

When administered to a patient, topical formulations are envisaged, andmay e.g. be convenient in relation to MRSA. Ointments, creams, wetwipes, sprays, and eye drops are alternative and illustrative ways ofadministering the compound according to the invention. In oneembodiment, the compound according to the invention is administered bycatheter to the bladder of the patient.

In one embodiment, the pharmaceutical composition comprising a compoundaccording to the invention is prepared for nasal application, e.g.against Staphylococcus aureus.

The bacterial agent causing the bacterial infection may be aGram-positive bacterial agent selected from the group comprising, butnot limited to, Staphylococcus spp, Streptococci, Enterococcus spp,Leuconostoc spp, Corynebacterium spp, Arcanobacteria spp, Trueperellaspp, Rhodococcus spp, Bacillus spp, Anaerobic Cocci, AnaerobicGram-Positive Nonsporulating Bacilli, Actinomyces spp, Clostridium spp,Nocardia spp, Erysipelothrix spp, Listeria spp, Kytococcus spp,Mycoplasma spp, Ureaplasma spp, and Mycobacterium spp.

In one embodiment, the bacterial agent is Gram-positive and is selectedfrom the group comprising, but not limited to, Staphylococcus spp.Examples of Staphylococcus spp include Staphylococcus epidermidis,Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcussaprophytics, Staphylococcus auricularis, Staphylococcus capitis,Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus cohnii,Staphylococcus hominis, Staphylococcus pasteuri, Staphylococcuspettenkoferi, Staphylococcus pulvereri, Staphylococcus saccharolyticus,Staphylococcus simulans, Staphylococcus schieiferi, Staphylococcuswarneri, Staphylococcus xyiosus, Staphylococcus arlettae, Staphylococcuscaseolyticus, Staphylococcus chromogenes, Staphylococcus condimenti,Staphylococcus delphini, Staphylococcus equorum, Staphylococcus felis,Staphylococcus fleurettii, Staphylococcus gallinarum, Staphylococcushyicus, Staphylococcus intermedius, Staphylococcus kloosii,Staphylococcus lentus, Staphylococcus lutrae, Staphylococcus muscae,Staphylococcus nepalensis, Staphylococcus piscifermentans,Staphylococcus pseudintermedius, Staphylococcus sciuri, Staphylococcussimiae, Staphylococcus succinus, and Staphylococcus vitulinus.

In another embodiment, the bacterial agent is Gram-positive and isselected from the group comprising, but not limited to, Streptococcusspp. Examples of Streptococcus spp include Streptococcus agalactiae,Streptococcus alactolyticus, Streptococcus anginosus, Streptococcuscanis, Streptococcus constellatus, Streptococcus cricetus, Streptococcuscristatus, Streptococcus downei, Streptococcus dysgalactiae subsp.dysgalactiae, Streptococcus dysgalactiae subsp. equisimilis,Streptococcus equi subsp. equi, Streptococcus equi subsp. zooepidemicus,Streptococcus ferus, Streptococcus gallolyticus subsp. gallolyticus(formerly Streptococcus bovis biotype i), Streptococcus gallolyticussubsp. pasteurianus (formerly Streptococcus bovis biotype ii/2),Streptococcus gordonii, Streptococcus hyointestinalis, Streptococcushyovaginalis, Streptococcus infantarius, Streptococcus infantarius subspinfantarius, Streptococcus infantis, Streptococcus iniae, Streptococcusintermedius, Streptococcus lutetiensis (formerly Streptococcus bovisbiotype ii.1), Streptococcus macaccae, Streptococcus mitis,Streptococcus mutans, Streptococcus oralis, Streptococcus orisratti,Streptococcus parasanguinis, Streptococcus peroris, Streptococcuspneumoniae, Streptococcus porcinus, Streptococcus pseudintermedius,Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius,Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis,Streptococcus thermophilus, Streptococcus vestibularis, andNutritionally Variant (Deficient) Streptococci (Abiotrophia defectiva,Granulicatella adiacens, Granulicatella elegans, and Granulicatellapara-adiacens) and related species such as Rothia mucilaginosa (formerlyStomatococcus mucilaginosus) and Pediococcus.

In another embodiment, the bacterial agent is Gram-positive and selectedfrom the group comprising, but not limited to, Enterococcus spp.Examples of Enterococcus spp include Enterococcus faecalis, Enterococcusfaecium, Enterococcus gallinarum, Enterococcus durans, Enterococcusavium, Enterococcus raffinosus, Enterococcus pallens, Enterococcusgilvus, Enterococcus cecorum, Enterococcus malodoratus, Enterococcusitalicus, Enterococcus sanguinicola, Enterococcus mundtii, Enterococcuscasseliflavus/flavescens, Enterococcus dispar, Enterococcus hirae,Enterococcus pseudoavium, and Enterococcus bovis.

In another embodiment, the bacterial agent is Gram-positive and selectedfrom the group comprising, but not limited to, Leuconostoc spp. Examplesof Leuconostoc spp include Leuconostoc mesenteroides, Leuconostocpseudomesenteroides, Leuconostoc paramesenteroides, Leuconostoc citreum,and Leuconostoc lactis.

In another preferred embodiment, the bacterial agent is Staphylococcusaureus, and/or strains known to be resistant such as MRSA or mupirocinresistant strain.

The bacterial agent causing the bacterial infection may be aGram-negative bacterial agent selected from the group comprising, butnot limited to, Neisseria spp, Moraxella spp, Escherichia spp,Klebsiella spp, Proteus spp, Pseudomonas spp, Salmonella spp, Shigellaspp, Campylobacter spp, Helicobacter spp, Bacteroides spp, Yersinia spp,Vibrio spp, Bordatella spp and Legionella spp. A specific example of aGram-negative bacterial agent causing the infection is Acinetobacterbaumannii, as appears from the Experimental part below, see e.g. Table2.

The bacterial agent causing the bacterial infection might be anaerobicbacteria, a spirochete or other form of bacteria including Clostridiumspp, Borrelia spp, Chlamydia spp and Mycoplasma spp.

In another preferred embodiment, the bacterial agents are resistant toone or several antibiotics in present use and may be selected from, butnot limited to, Clostridium difficile, carbapenem-resistantEnterobacteriaceae, Neisseria gonorrhoeae, multidrug-resistantAcinetobacter, drug-resistant Campylobacter, extended spectrumbeta-lactamase producing Enterobacteriaceae, multidrug-resistantPseudomonas aeruginosa, drug-resistant Salmonella, drug-resistantShigella and drug-resistant Tuberculosis.

In another preferred embodiment, the bacterial agent is resistant to acompound selected from the group comprising: one or more ofaminoglycosides (for example gentamicin); ansamycins {for examplerifampicin); anti-MRSA cephalosporins (for example ceftaroline);anti-staphylococcal β-lactams (or cephamycins) (for example oxacillin orcefoxitin); carbapenems (for example ertapenem, imipenem, meropenem ordoripenem); non-extended spectrum cephalosporins; 1 st and 2ndgeneration cephalosporins (for example cefazolin or cefuroxime);extended-spectrum cephalosporins; 3rd and 4th generation cephalosporins(for example cefotaxime or ceftriaxone); cephamycins (for examplecefoxitin or cefotetan); fluoroquinolones (for example ciprofloxacin ormoxifloxacin); folate pathway inhibitors (for exampletrimethoprim-sulphamethoxazole); fucidanes (for example fusidic acid);glycopeptides (for example vancomycin, teicoplanin or telavancin);glycylcyclines (for example tigecycline); lincosamides (for exampleclindamycin); lipopeptides (for example daptomycin); macrolides (forexample erythromycin); monoxycarbolic acids, such as mupirocin;oxazolidinones (for example linezolid or tedizolid); phenicols (forexample chloramphenicol); phosphonic acids (for example fosfomycin);streptogramins (for example quinupristin-dalfopristin); andtetracyclines (for example tetracycline, doxycycline or minocycline).

In another preferred embodiment, the bacterial agent is Streptococcuspneumoniae. The Streptococcus pneumoniae may be a strain that isresistant to one or more of β-lactams and macrolides.

In another preferred embodiment, the bacterial agent is Streptococcuspyogenes.

In another most preferred embodiment, the bacterial agent isStreptococcus agalactiae.

In another most preferred embodiment, the bacterial agent is eitherEnterococcus faecium or Enterococcus faecalis. The Enterococcus faeciumor Enterococcus faecalis may be a strain that is resistant toaminoglycosides (for example gentamicin (high level) or streptomycin(for example streptomycin (high level)); carbapenems (for exampleimipenem, meropenem or doripenem); fluoroquinolones (for exampleciprofloxacin, levofloxacin or moxifloxacin); glycopeptides (for examplevancomycin or teicoplanin); glycylcyclines (for example tigecycline);lipopeptides (for example daptomycin); oxazolidinones (for examplelinezolid); penicillins (for example ampiciilin); streptogramins (forexample quinupristin-dalfopristin); tetracycline (for exampledoxycycline or minocycline).

In another embodiment, the bacterial agent is Clostridium difficile.

The bacterial infection in the subject may cause a disease selected fromthe group comprising, but not limited to, nosocomial pneumonia caused byStaphylococcus aureus (MDR, XDR, PDR or methicillin-susceptible or-resistant strains), or invasive pneumococcal diseases such aspneumonia, bronchitis, acute sinusitis, otitis media, conjunctivitis,meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis,endocarditis, peritonitis, pericarditis, cellulitis, and brain abscesscaused by Streptococcus pneumoniae (including multi-drug resistantstrains [MDRSP] such as those resistant to β-lactams and macrolides),complicated skin and skin structure infections, including diabetic footinfections, with or without concomitant osteomyelitis, caused byStaphylococcus aureus (methicillin-susceptible and -resistant strains),Streptococcus pyogenes, or Streptococcus agalactiae, uncomplicated skinand skin structure infections caused by Staphylococcus aureus(methicillin-susceptible and -resistant strains) or Streptococcuspyogenes, community-acquired pneumonia caused by Streptococcuspneumoniae (including multi-drug resistant strains [MDRSP], includingcases with concurrent bacteraemia, or Staphylococcus aureus(methicillin-susceptible and -resistant strains) and Staphylococcusaureus bloodstream Infections (bacteraemia), including those withright-sided infective endocarditis, caused by methicillin-susceptibleand methicillin-resistant isolates, vancomycin-resistant enterococcusinfections, including cases with concurrent bacteraemia, and treatmentof Clostridium d/f/c//e-associated diarrhea (CDAD).

In another preferred embodiment, a compound of the invention, or atherapeutically acceptable salt thereof, is administered together with acompound or agent that removes or substantially removes or reduces theintegrity of the cell wall of the bacterial agent. As an example, thecompound is selected from the group consisting of: P lactams,fosfomycin, lysozyme, polymyxins and chelating agents such asethylenediaminetetraacetic acid (EDTA). As an example, the agent is animmunological agent (such as an antibody or vaccine) that reduces theintegrity of the cell wall. In one preferred embodiment, the compound,or a therapeutically acceptable salt thereof, is administered togetherwith a compound that removes or substantially removes or weakens theintegrity of the outer cell wall of a Gram-negative bacterial agent.

According to another aspect of the invention, there is provided anantibacterial pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of Formula I, or a therapeuticallyacceptable salt thereof. Preferably, the composition is an antibacterialpharmaceutical composition.

According to another aspect of the invention, there is provided anantibacterial veterinary composition comprising a therapeuticallyeffective amount of a compound of Formula I, or a therapeuticallyacceptable salt thereof. Preferably, the composition is ananti-bacterial veterinary composition, for topical or oral use.

The pharmaceutical composition may optionally include a pharmaceuticallyacceptable excipient or carrier. The veterinary composition mayoptionally include a veterinary acceptable excipient or carrier.

In another embodiment, the pharmaceutical or veterinary compositioncomprises impurities, wherein the quantity of impurities as a percentageof the total weight of the composition is selected from the groupconsisting of: less than 20% impurities (by total weight of thecomposition); less than 15% impurities; less than 10% impurities; lessthan 8% impurities; less than 5% impurities; less than 4% impurities;less than 3% impurities; less than 2% impurities; less than 1%impurities; less than 0.5% impurities; less than 0.1% impurities. In oneembodiment, the pharmaceutical or veterinary composition comprisesmicrobial impurities or secondary metabolites, wherein the quantity ofmicrobial impurities as a percentage of the total weight of thecomposition is selected from the group consisting of: less than 5%; lessthan 4%; less than 3%; less than 2%; less than 1%; less than 0.5%; lessthan 0.1%; less than 0.01%; less than 0.001%. In one embodiment, thepharmaceutical or veterinary composition is sterile and stored in asealed and sterile container. In one embodiment, the pharmaceutical orveterinary composition contains no detectable level of microbialcontamination.

The pharmaceutical or veterinary composition of the invention maycomprise a further antimicrobial agent. The further antimicrobial agentmay be an antifungal agent or antibacterial agent. The method oftreating or preventing a bacterial infection or colonisation in asubject, may also comprise the administration of a compound of theinvention with a further antimicrobial agent.

In one embodiment, the pharmaceutical composition comprises a compoundaccording to the invention in combination with mupirocin. Such acomposition may e.g. be for topical application, for example anointment, or any other form as discussed elsewhere in this application.

The pharmaceutical or veterinary composition of the invention maycomprise more than one compound of the invention, such as a combinationof compounds. The method of treating or preventing a bacterial infectionor colonisation in a subject may also comprise the administration ofmore than one compound of the invention.

In one embodiment, the antifungal agent is selected from the groupcomprising, but not limited to naturally occurring agents includingEchinocandins (Anidulafungin, Caspofungin, Micafungin), Polyenes(Amphotericin B, Candicidin, Filipin, Fungichromin (Pentamycin),Hachimycin, Hamycin, Lucensomycin, Mepartricin, Natamycin, Nystatin,Pecilocin, Perimycin), and other naturally occurring antifungal agentsincluding Griseofulvin, Oligomycins, Pyrrolnitrin, Siccanin, andViridin. The antifungal agent may be a synthetic compound selected fromthe group comprising, but not limited to Allylamines (Butenafine,Naftifine, Terbinafine) Imidazoles (Bifonazole, Butoconazole,Chlormidazole, Climbazole, Croconazole (Cloconazole), Clotrimazole,Eberconazole, Econazole, Enilconazole, Fenticonazole, Flutrimazole,Fosfluconazole, Isoconazole, Ketoconazole, Lanoconazole, Luliconazole,Miconazole, Neticonazole, Omoconazole, Oxiconazole Nitrate, Parconazole,Sertaconazole, Sulconazole, Tioconazole), Thiocarba mates (Liranaftate,Tolciclate, Tolindate, Tolnaftate), Triazoles (Fluconazole,Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Saperconazole,Terconazole, Voriconazole), and other synthetic agents such asAcrisorcin, Amorolfine, Bromosalicylchloranilide(Bromochlorosalicylanilide), Buclosamide, Calcium Propionate,Chlorphenesin, Ciclopirox, Cloxyquin (Cloxiquine), Coparaffinate,Exalamide, Flucytosine, Haloprogin, Hexetidine, Loflucarban, Nifuratel,Nifuroxime, Piroctone, Potassium Iodide, Propionic Acid, Pyrithione,Salicylanilide, Sodium Parachlorobenzoate, Sodium Propionate,Sulbentine, Tenonitrozole, Triacetin, Trimetrexate, Undecylenic Acid(Undecenoic Acid), and Zinc Propionate.

The composition of the invention may comprise an antibiotic adjunctselected from the group comprising, but not limited to, β-LactamaseInhibitors (Avibactam, Clavulanic Acid, Sulbactam, Sultamicillin,Tazobactam), Renal Dipeptidase Inhibitors (Cilastatin), and RenalProtectant (Betamipron).

In one embodiment, the composition of the invention comprises a furtherantibiotic selected from the group comprising, but not limited to,

2,4-DIAMINOPYRIMIDINES, including Baquiloprim, Brodimoprim, Iclaprim,Ormetoprim, Pyrimethamine, Tetroxoprim, Trimethoprim;

AMINOCOUMARINS, including Novobiocin;

AMINOCYCLITOLS, including Spectinomycin;

AMINOGLYCOSIDES, including Amikacin, Apramycin, Arbekacin, Bekanamycin,Butirosin, Dibekacin, Dihydrostreptomycin, Etimicin, Fortimicins(Astromicin), Framycetin, Gentamicin, Hygromycin B, Isepamicin,Kanamycin, Micronomicin, Neomycin, Netilmicin, Paromomycin, Plazomicin,Ribostamycin, Sisomicin, Streptomycin, Tobramycin, Verdamicin;AMINOMETHYLCYCLINES, including Omadacycline;AMPHENICOLS, including Azidamfenicol, Chloramphenicol, Florfenicol,Thiamphenicol;ANSAMYCINS, including Rifabutin, Rifamide, Rifampin (Rifampicin),Rifamycin, Rifapentine, Rifaximin;ANTISEPTIC AGENTS, including Acridine derivatives (includingacriflavine, aminoacridine, ethacridine, proflavine), Bispyridines(including octenidine dihydrochloride), Brominated salicylanilides(including bromsalans), Chlorhexidine, Phenol derivatives (includingthymol and triclosan), Quarternary ammonium compounds (includingAlkyldimethylethylbenzyi Ammonium Chloride, benzalkonium chloride,cetylpyridinium chloride, benzethonium chloride, cetrimonium);ANTITUBERCULAR AGENTS, including Cycloserine, Delamanid, Ethambutol,Ethionamide, Isoniazid (Ftivazide), Morinamide, p-Aminosalicylic Acid(PAS), Protionamide, Pyrazinamide, Terizidone, Thioacetazone,Tiocarlide;ARSENICALS, including Arsanilic Acid, Roxarsone;BACTERIOCINS, including Nisin, Brilacidin (PMX-30063);β-LACTAM CARBACEPHEMS, including Loracarbef;β-LACTAM CARBAPENEMS, including Biapenem, Doripenem, Ertapenem,Faropenem, Imipenem, Meropenem, Panipenem, Razupenem, Ritipenem,Sulopenem, Tebipenem, Tomopenem;β-LACTAM CEPHALOSPORINS, including Cefacetrile, Cefaclor, Cefadroxil,Cefalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalothin,Cefamandole, Cefapirin, Cefatrizine, Cefazaflur, Cefazedone, Cefazolin,Cefcapene, Cefdinir, Cefditoren, Cefepime, Cefetamet, Cefixime,Cefmenoxime, Cefodizime, Cefonicid, Cefoperazone, Ceforanide, Cefose!is,Cefotaxime, Cefotiam, Cefovecin, Cefozopran, Cefpimizole, Cefpiramide,Cefpirome, Cefpodoxime, Cefprozil, Cefquinome, Cefradine, Cefroxadine,Cefsulodin, Ceftaroline, Ceftazidime, Cefteram, Ceftezole, Ceftibuten,Ceftiofur, Ceftizoxime, Ceftobiprole, Ceftolozane, Ceftradine,Ceftrezole, Ceftriaxone, Ceftroxadine, Cefuroxime, Cefuzonam,Pivcefalexin;β-LACTAM CEPHAMYCINS, including Cefbuperazone, Cefmetazole, Cefminox,Cefotetan, Cefoxitin; β-LACTAM MONOBACTAMS, including Aztreonam,Carumonam, Tigemonam;β-LACTAM OXACEPHEMS, including Flomoxef, Latamoxef, Moxalactam;β-LACTAM PENICILLINS, including Amdinocillin (Mecillinam), Amoxicillin,Ampicillin, Apalcillin, Aspoxicillin, Azidocillin, Azlociliin,Bacampicillin, Carbenicillin, Carindacillin, Ciclacillin, ClemizolePenicillin, Clometocillin, Cloxacillin, Cyclacillin, Dicloxacillin,Epicillin, Fenbenicillin, Floxacillin (Flucloxacillin), Hetacillin,Lenampicillin, Mecillinam, Metampicillin, Methicillin Sodium,Mezlocillin, Nafcillin, Oxacillin, Penamecillin, Penethamate Hydriodide,Penicillin G, Penicillin G Benzathine, Penicillin G Procaine, PenicillinN, Penicillin O, Penicillin V, Phenethicillin Potassium, Piperacillin,Pivampicillin, Pivmecillinam, Propicillin, Quinacillin, Sulbenicillin,Sultamicillin, Talampicillin, Temocillin, Ticarcillin;BICYCLOMYCINS, including Bicozamycin;BORON CONTAINING ANTIBACTERIAL AGENTS, including AN3365(aminomethylbenzoxaboroles), GSK2251052 (leucyl-tRNA synthetaseinhibitors);CYCLIC ESTERS, including Fosfomycin;EFFLUX PUMP INHIBITORS;FATTY ACID SYNTHESIS INHIBITORS (FabI), AFN-1252, MUT056399, FAB-001FLUOROQUINOLONES, including Avarofloxacin, Balofloxacin, Besifloxacin,Chinfloxacin, Cinoxacin, Ciprofloxacin, Clinafloxacin, Danofloxacin,Delafloxacin, Difloxacin, Enoxacin, Enrofloxacin, Finafloxacin,Fleroxacin, Flumequine, Garenoxacin, Gatifloxacin, Gemifloxacin,Grepafloxacin, Ibafloxacin, Levofloxacin, Lomefioxacin, Marbofloxacin,Miloxacin, Moxifloxacin, Nadifloxacin, Norfloxacin, Ofloxacin,Orbifloxacin, Pazufloxacin, Pefloxacin, Pradofloxacin, Prulifloxacin,Rosoxacin, Rufloxacin, Sarafloxacin, Sitafloxacin, Sparfloxacin,Temafloxacin, Tosufloxacin, Trovafloxacin, Zabofloxacin;FUSIDANES, including Fusidic Acid;GLYCOLIPODEPSIPEPTIDE, including Ramoplanin;GLYCOPEPTIDES, including Avoparcin, Dalbavancin, Norvancomycin,Oritavancin, Teicoplanin, Telavancin, Vancomycin;GLYCOPHOSPHOLIPIDS, including Bambermycins (bambermycin, moenomycins,flavophospholipol);GLYCYLCYCLINES, including Tigecycline;HYBRIDS, Cadazolid (Oxazolidinone-quinolone), TD-1792(glycopeptide-cephalosporin);LINCOSAMIDES, including Clindamycin, Lincomycin, Pirlimycin;LIPOPEPTIDES, including Daptomycin, Surotomycin;MACROLIDES, including Azithromycin, Carbomycin, Cethromycin,Clarithromycin, Dirithromycin, Erythromycin, Fidaxomicin,Flurithromycin, Gamithromycin, Josamycin, Kitasamycin, Leucomycin,Meleumycin, Midecamycins, Miokamycin, Mirosamycin, Oleandomycin,Primycin, Rokitamycin, Rosaramicin, Roxithromycin, Sedecamycin,Solithromycin, Spiramycin, Telithromycin, Terdecamycin, Tildipirosin,Tilmicosin, Troleandomycin, Tulathromycin, Tylosin, Tylvalosin;NITROFURANS, including Furaltadone, Furazidin, Furazolidone, FurazoliumChloride, Nifuratel, Nifurfoline, Nifuroxazide, Nifurpirinol,Nifurtoinol, Nifurzide, Nitrofural, Nitrofurantoin, Nitrofurazone;NITROIMIDAZOLES, including Dimetridazole, Metronidazole, Ornidazole,Ronidazole, Secnidazole, Tinidazole;OLIGOSACCHARIDES, including Avilamycin, Everninomicin;OTHER ANTIBACTERIAL AGENTS, including Auriclosene, Chloroxine,Chlorquinaldol, Clioquinol, Clofoctol, Halquinol, Lotilibcin, MandelicAcid, Methenamine (hexamine), Nitazole, Nitroxoline, Perchlozone,Taurolidine, Thenoic Acid, Xibornol;OXAZOLID1NONES, including Eperezolid, Linezolid, Posizolid, Radezolid,Sutezolid, Tedizolid (Torezolid);PEPTIDE DEFORMYLASE INHIBITORS, including GSK1322322;PEPTIDES, including Omiganan, Pexiganan;PLEUROMUTILINS, including Retapamulin, Tiamulin, Valnemulin;POLYETHER IONOPHORES, including Laidlomycin, Lasalocid, Maduramicin,Monensin, Narasin, Salinomycin, Semduramicin;POLYMYXINS, including Colistin, Polymyxin B;POLYPEPTIDES, including Amphomycin, Bacitracin, Capreomycin,Enduracidin, Enramycin, Enviomycin, Fusafungine, Gramicidin(s),Iseganan, Magainins, Nosiheptide, Ristocetin, Thiostrepton,Tuberactinomycin, Tyrocidine, Tyrothricin, Viomycin;PSEUDOMONIC ACIDS, including Mupirocin;QUINOLONES, including Nalidixic Acid, Nemonoxacin, Oxolinic Acid,Ozenoxacin, Pipemidic Acid, Piromidic Acid;QUINOXALINES, including Carbadox, Olaquindox;RIMINOFENAZINES, including Clofazimine;STATINS, including Atorvastatin, Fluvastatin, Lovastatin, Mevastatin,Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin;STREPTOGRAMINS, including Dalfopristin, Flopristin, Linopnstin,Pristinamycin, Quinupristin, Virginiamycin;STREPTOTHRICINS, including Nourseothricin;SULFONAMIDES, including Acetyl Sulfamethoxypyrazine, Chloramine-B,Chloramine-T, Dichloramine T, Formosulfathiazole, Mafenide,N4-Sulfanilylsulfanilamide, Noprylsulfamide, N-Sulfanilyl-3,4-xylamide,Ormaosulfathiazole, Phthalylsulfacetamide, Phthalylsulfathiazole,Salazosulfadimidine, Succinylsulfathiazole, Sulfabenzamide,Sulfacarbamide, Sulfacetamide, Sulfachlorpyridazine, Sulfachrysoidine,Sulfaclozine, Sulfacytine, Sulfadiazine, Sulfadicramide,Sulfadimethoxine, Sulfadimidine, Sulfadoxine, Sulfaethidole,Sulfaguanidine, Sulfaguanole, Sulfalene, Sulfaloxic Acid, Sulfamerazine,Sulfameter, Sulfamethazine, Sulfamethizole, Sulfamethomidine,Sulfamethoxazole, Sulfamethoxypyridazine, Sulfamethylthiazole,Sulfametopyrazine, Sulfametrole, Sulfamidochrysoidine,Sulfamonomethoxine, Sulfamoxole, Sulfanilamide, Sulfanilylurea,Sulfaperine, Sulfaphenazole, Sulfaproxyline, Sulfapyrazine,Sulfapyridine, Sulfaquinoxaline, Sulfathiazole, Sulfathiourea,Sulfatroxazole, Sulfisomidine, Sulfisoxazole (Sulfafurazole);SULFONES, including Acediasulfone, Dapsone, Glucosulfone Sodium,p-Sulfanilylbenzylamine, Succisulfone, Sulfanilic Acid, SulfoxoneSodium, Thiazolsulfone;TETRACYCLINES, including Chlortetracycline, Clomocycline,Demeclocycline, Doxycycline, Eravacycline, Guamecycline, Lymecycline,Meclocycline, Methacycline, Minocycline, Oxytetracycline,Penimepicycline, Pipacycline, Rolitetracycline, Sarecycline, andTetracycline.

The composition of the invention may further comprise an excipientselected from the group comprising, but not limited to, binders andcompression aids, coatings and films, colouring agents diluents andvehicles disintegrants, emulsifying and solubilising agents, flavoursand sweeteners, repellents, glidants and lubricants, plasticisers,preservatives, propellants, solvents, stabilisers, suspending agents andviscosity enhancers.

According to a further aspect of the invention, there is provided amedical device when used in a method of treating or preventing abacterial infection in the subject.

According to further aspect of the invention, there is provided amedical device comprising the composition of the invention. Thecomposition of the invention may be any slow release form, and/or in theform of a coating of the medical device.

The medical device may be in a form selected from the group comprising:an implant, a plaster, a bandage, and other dressing applied to abacterial infection in a subject.

According to further aspect of the invention, there is provided a methodof killing bacteria, the method including the step of contacting thebacteria with a compound of the invention, or a therapeuticallyacceptable salt thereof. As discussed elsewhere in the presentapplication, administration may e.g. be by creme, ointment, eye drops orby catheter to the bladder of the patient.

According to further aspect of the invention, there is provided the useof a compound of the invention, or a therapeutically acceptable saltthereof, to kill bacteria, said use comprising the step of contactingthe bacteria with a compound of the invention, or a therapeuticallyacceptable salt thereof.

DESCRIPTION OF EMBODIMENTS

General

Before describing the present invention in detail, it is to beunderstood that the invention is not limited to particular exemplifiedmethods or compositions disclosed herein. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments of the invention only, and is not intended to belimiting.

All publications referred to herein, including patents or patentapplications, are incorporated by reference in their entirety. However,applications that are mentioned herein are referred to simply for thepurpose of describing and disclosing the procedures, protocols, andreagents referred to in the publication which may have been used inconnection with the invention. The citation of any publications referredto herein is not to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

In addition, the carrying out of the present invention makes use of,unless otherwise indicated, conventional microbiological techniqueswithin the skill of the art. Such conventional techniques are known tothe skilled worker.

As used herein, and in the appended claims, the singular forms “a”,“an”, and “the” include the plural unless the context clearly indicatesotherwise.

Unless otherwise indicated, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any materialsand methods similar to, or equivalent to, those described herein may beused to carry out the present invention, the preferred materials andmethods are herein described.

The invention described herein may include one or more ranges of values(e.g. size, concentration, dose etc). A range of values will beunderstood to include all values within the range, including the valuesdefining the range, and values adjacent to the range that lead to thesame or substantially the same outcome as the values immediatelyadjacent to that value which define the boundary of the range.

The pharmaceutical or veterinary compositions of the invention may beadministered in a variety of unit dosages depending on the method ofadministration, target site, physiological state of the patient, andother medicaments administered. For example, unit dosage form suitablefor oral administration include solid dosage forms such as powder,tablets, pills, and capsules, and liquid dosage forms, such as elixirs,syrups, solutions and suspensions. The active ingredients may also beadministered parenterally in sterile liquid dosage forms. Gelatincapsules may contain the active ingredient and inactive ingredients suchas powder carriers, glucose, lactose, sucrose, mannitol, starch,cellulose or cellulose derivatives, magnesium stearate, stearic acid,sodium saccharin, talcum, magnesium carbonate, and the like. Topicalforms were discussed above and are included as an alternative in allaspects of the present invention.

The phrase “therapeutically effective amount” as used herein refers toan amount sufficient to inhibit bacterial growth associated with abacterial infection or colonisation. That is, reference to theadministration of the therapeutically effective amount of a compound ofFormula I according to the methods or compositions of the inventionrefers to a therapeutic effect in which substantial bacteriocidal orbacteriostatic activity causes a substantial inhibition of bacterialinfection. The term “therapeutically effective amount” as used herein,refers to a sufficient amount of the composition to provide the desiredbiological, therapeutic, and/or prophylactic result. The desired resultsinclude elimination of bacterial infection or colonisation or reductionand/or alleviation of the signs, symptoms, or causes of a disease, orany other desired alteration of a biological system. An effective amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation. In relation to a pharmaceutical orveterinary composition, effective amounts can be dosages that arerecommended in the modulation of a diseased state or signs or symptomsthereof. Effective amounts differ depending on the composition used andthe route of administration employed. Effective amounts are routinelyoptimized taking into consideration pharmacokinetic and pharmacodynamiccharacteristics as well as various factors of a particular patient, suchas age, weight, gender, etc and the area affected by disease or diseasecausing microbes.

As referred to herein, the terms “treatment” or “treating” refers to thefull or partial removal of the symptoms and signs of the condition aswell as any trace or sign of the causing bacteria. For example, in thetreatment of a bacterial infection or colonisation, the treatmentcompletely or partially removes the signs of the infection as well asany bacteria as such. Preferably in the treatment of infection, thetreatment reduces or eliminates the infecting bacterial pathogen leadingto microbial cure, and prevention of transmission of any seriousdevelopment of the infection. The term “elimination” refers herein tothe total removal of all signs and trace of an infection.

As referred to herein, the term “bacteria” refers to members of a largedomain of prokaryotic microorganisms. Typically a few micrometers inlength, bacteria have a number of shapes, ranging from spheres to rodsand spirals and can be present as individual cells or present in linearchains or clusters of variable numbers and shape. Preferably the terms“bacteria” and its adjective “bacterial” refer to bacteria such as theGram-positive Staphylococcus spp, Streptocccus spp, Bacillus spp,Enferococcus spp, Listeria spp, and anaerobic bacteria; The terms mayrefer to an antibiotic-sensitive strain or an antibiotic-resistantstrain. In a preferred embodiment, the terms refer to MRSA or MRSP. Inanother preferred embodiment, the terms refer to MDR Staphylococcus spp,Streptococcus spp, Enterococcus spp, Clostridium difficile. The term mayalso refer to Gram-negative bacteria such as Enterobaceriaceae spp,Klebsiella spp, Neisseria spp, Acinetobacter spp, Campylobacter spp,Salmonella spp, Shigella spp, Pseudomonas spp.

Referred to herein, the term “methicillin-resistant bacteria” (such asmethicillin-resistant Staphylococcus) refers a bacteria isolate thatdemonstrates resistance at any dose to all 3-lactams includingpenicillins, carbapenems and first to fourth generation cephalosporins,but not to the fifth generation anti-MRSA cephalosporins (for exampleceftaroline). Multidrug-resistant (MDR) is defined as acquirednon-susceptibility to at least one agent in three or more antimicrobialcategories, extensively drug-resistant (XDR) is defined asnon-susceptibility to at least one agent in all but two or fewerantimicrobial categories (i.e. bacterial isolates remain susceptible toonly one or two categories) and pandrug-resistant (PDR) is defined asnon-susceptibility to all agents in all antimicrobial categoriescurrently available.

An example of susceptible, MDR, XDR and PDR bacteria includes thefollowing. Wild type, antibacterial unexposed isolates of Staphylococcusaureus that are likely to be susceptible to all of the followingantibacterial categories (and agents); aminoglycosides (for examplegentamicin); ansamycins (for example rifampicin); anti-MRSAcephalosporins (for example ceftaroline); anti-staphylococcal β-lactams(or cephamycins) (for example oxacillin or cefoxitin); carbapenems (forexample ertapenem, imipenem, meropenem or doripenem); non-extendedspectrum cephalosporins; 1 st and 2nd generation cephalosporins (forexample cefazolin or cefuroxime); extended-spectrum cephalosporins; 3rdand 4th generation cephalosporins (for example cefotaxime orceftriaxone); cephamycins (for example cefoxitin or cefotetan);fluoroquinolones (for example ciprofloxacin or moxifloxacin); folatepathway inhibitors (for example trimethoprim-sulphamethoxazole);fucidanes (for example fusidic acid); glycopeptides (for examplevancomycin, teicoplanin or telavancin); glycylcyclines (for exampletigecycline); lincosamides (for example clindamycin); lipopeptides (forexample daptomycin); macrolides (for example erythromycin);oxazolidinones (for example linezolid or tedizolid); phenicols (forexample chloramphenicol); phosphonic acids (for example fosfomycin);streptogramins (for example quinupristin-dalfopristin; and tetracyclines(for example tetracycline, doxycycline or minocycline). Isolates thatare non-susceptible to more than one agent in more than threeantimicrobial categories are classified as MDR (all MRSA, for example,meet the definition of MDR). Isolates that are non-susceptible to morethan one agent in all but one or two antimicrobial categories areclassified as XDR. Isolates that are non-susceptible to all listedantibacterial agents are PDR.

The use of compounds according to the invention, as defined by FormulaI, avoids the use of other antibiotics and thus resistance developmentto such antibiotics.

Pharmaceutically and veterinary acceptable salts include salts whichretain the biological effectiveness and properties of the compounds ofthe present disclosure and which are not biologically or otherwiseundesirable. In many cases, the compounds disclosed herein are capableof forming acid and/or base salts by virtue of the presence of aminoand/or carboxyl groups or groups similar thereto. Acceptable baseaddition salts can be prepared from inorganic and organic bases. Saltsderived from inorganic bases, include by way of example only, sodium,potassium, lithium, ammonium, calcium and magnesium salts. Salts derivedfrom organic bases include, but are not limited to, salts of primary,secondary and tertiary amines, such as by way of example only, alkylamines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri (substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amines,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amines, trisubstituted cycloalkenyl amines,aryl amines, diary’ amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group.

Pharmaceutically and veterinary acceptable acid addition salts may beprepared from inorganic and organic acids. The inorganic acids that canbe used include, by way of example only, hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, and the like. Theorganic acids that can be used include, by way of example only, aceticacid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malicacid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like.

The pharmaceutically or veterinary acceptable salts of the compoundsuseful in the present disclosure can be synthesized from the parentcompound, which contains a basic or acidic moiety, by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, non-aqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences. 17thed., Mack Publishing Company, Easton, Pa. (1985), p. 1418, thedisclosure of which is hereby incorporated by reference. Examples ofsuch acceptable salts are the iodide, acetate, phenyl acetate,trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methyl benzoate,o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate,phenylbutyrate, γ-hydroxybutyrate, β-hydroxybutyrate, butyne-I,4-dioate,hexyne-I,4-dioate, hexyne-I,6-dioate, caproate, caprylate, chloride,cinnamate, citrate, decanoate, formate, fumarate, glycollate,heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate,malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate,oxalate, phthalate, terephthalate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate,propionate, phenylpropionate, salicylate, sebacate, succinate, suberate,sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate,benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate,propanesu!fonate, ethanesulfonate, 2-hydroxyethanesulfonate,merhanesulfonate, naphthalene-I-sulfonate, naphthalene-2-sulfonate,p-toluenesulfonate, xylenesulfonate, tartarate, and the like.

The present invention also includes isotope-labelled compounds offormula I or any subgroup of formula I, wherein one or more of the atomsis replaced by an isotope of that atom, i.e. an atom having the sameatomic number as, but an atomic mass different from, the one(s)typically found in nature. Examples of isotopes that may be incorporatedinto the compounds of formula I or any subgroup of formula I, includebut are not limited to isotopes of hydrogen, such as ²H and ³H (alsodenoted D for deuterium and T for tritium, respectively), carbon, suchas ¹¹C, ¹³C and ¹⁴C, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O,¹⁷O and ¹⁸O, phosphorus, such as ³¹P and ³²P, sulfur, such as ³⁵S,fluorine, such as ¹⁸F, chlorine, such as ³⁶Cl, bromine such as ⁷⁵Br,⁷⁶Br, ⁷⁷Br and ⁸²Br, and iodine, such as ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. Thechoice of isotope included in an isotope-labelled compound will dependon the specific application of that compound. For example, for drug orsubstrate tissue distribution assays, compounds wherein a radioactiveisotope such as ³H or ¹⁴C is incorporated will generally be most useful.For radio-imaging applications, for example positron emission tomography(PET) a positron emitting isotope such as ¹¹C, ¹⁸F, ¹³N or ¹⁵O will beuseful. The incorporation of a heavier isotope, such as deuterium, i.e.²H, may provide greater metabolic stability to a compound of formula Ior any subgroup of formula I, which may result in, for example, anincreased in vivo half-life of the compound or reduced dosagerequirements.

Isotope-labelled compounds of formula I or any subgroup of formula I canbe prepared by processes analogous to those described in the Schemesand/or Examples herein below by using the appropriate isotope-labelledreagent or starting material instead of the correspondingnon-isotope-labelled reagent or starting material, or by conventionaltechniques known to those skilled in the art.

The compounds of the invention can have one or several chiral centersand may exist and be isolated in optically active and racemic forms.Some compounds may exhibit polymorphism. It is to be understood that anyracemic, optically active, diastereomeric, polymorphic or stereoisomericform or mixtures thereof, of a compound provided herein is within thescope of this invention. The absolute configuration of such compoundscan be determined using methods known in the art such as, for example,X-ray diffraction or NMR and/or implication from starting materials ofknown stereochemistry and/or stereoselective synthesis methods.Pharmaceutical compositions in accordance with the invention willpreferably comprise substantially stereoisomerically pure preparationsof the indicated stereoisomer.

Pure stereoisomeric forms of the compounds and intermediates asmentioned herein are defined as isomers substantially free of otherenantiomeric or diastereomeric forms of the same basic molecularstructure of said compounds or intermediates. In particular, the term“stereoisomerically pure” concerns compounds or intermediates having astereoisomeric excess of at least 80% (i.e. minimum 90% of one isomerand maximum 10% of the other possible isomers) up to a stereoisomericexcess of 100% (i.e. 100% of one isomer and none of the other), more inparticular, compounds or intermediates having a stereoisomeric excess of90% up to 100%, even more in particular having a stereoisomeric excessof 94% up to 100% and most in particular having a stereoisomeric excessof 97% up to 100%. The terms “enantiomerically pure” and“diastereomerically pure” should be understood in a similar way, butthen having regard to the enantiomeric excess, and the diastereomericexcess, respectively, of the mixture in question.

Pure stereoisomeric forms of the compounds and intermediates of thisinvention may be obtained by using procedures well known in the art. Forinstance, enantiomers may be separated from each other by resolution ofthe racemic mixture, i.e. formation of a diastereomeric salt effected byreaction with an optically active acid or base followed by selectivecrystallization of the formed diastereomeric salt. Examples of suchacids are tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acidand camphorsulfonic acid. Alternatively, enantiomers may be separated bychromatographic techniques using chiral stationary phases. Purestereochemically isomeric forms may also be obtained by synthesis fromstereochemically pure forms of the appropriate starting materials,provided that the reaction occurs stereospecifically, by chiralsynthesis or by utilisation of a chiral auxiliary. If a specificstereoisomer is desired, the preparation of that compound is preferablyperformed using stereospecific methods. These methods willadvantageously employ enantiomerically pure starting materials.

Diastereomeric racemates of the compounds of the invention can beseparated by conventional methods. Appropriate physical separationmethods that may advantageously be employed are, for example, selectivecrystallization and chromatography, e.g. column chromatography.

The terms and expressions used herein throughout the abstract,specification and claims shall be interpreted as defined below unlessotherwise indicated. The meaning of each term is independent at eachoccurrence. These definitions apply regardless of whether a term is usedby itself or in combination with other terms, unless otherwiseindicated. A term or expression used herein which is not explicitlydefined, shall be interpreted as having its ordinary meaning used in theart. Chemical names, common names, and chemical structures may be usedinterchangeably to describe the same structure. If a chemical compoundis referred to using both a chemical structure and a chemical name andan ambiguity exists between the structure and the name, the structurepredominates.

“C_(m)-C_(n)alkyl” on its own or in composite expressions such asC_(m)-C_(n)haloalkyl, C_(m)-C_(n)alkylcarbonyl, C_(m)-C_(n)alkylamine,etc. represents a straight or branched aliphatic hydrocarbon radicalhaving the number of carbon atoms designated, e.g. C₁-C₄alkyl means analkyl radical having from 1 to 4 carbon atoms. C₁-C₆alkyl has acorresponding meaning, including also all straight and branched chainisomers of pentyl and hexyl. Preferred alkyl radicals for use in thepresent invention are C₁-C₆alkyl, including methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-buty, tert-butyl, n-pentyl andn-hexyl, especially C₁-C₄alkyl such as methyl, ethyl, n-propyl,isopropyl, tert-butyl, n-butyl and isobutyl. Methyl and isopropyl aretypically preferred. An alkyl group may be unsubstituted or substitutedby one or more substituents which may be the same or different, eachsubstituent being independently selected from the group consisting ofhalo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy,C_(m)-C_(n)alkoxy, —O-aryl, —C_(m)-C_(n)alkoxyC_(m)-C_(n)alkyl,C_(m)-C_(n)alkylthio, —NH₂, —NH(C_(m)-C_(n)alkyl),—N(C_(m)-C_(n)alkyl)₂, —NH(cycloalkyl), —OC(O)alkyl, —O—C(O)aryl,—O—C(O)cycloalkyl, —C(O)OH and —C(O)OC_(m)-C_(n)alkyl. It is generallypreferred that the alkyl group is unsubstituted, unless otherwiseindicated.

“C₂-C_(n)alkenyl” represents a straight or branched aliphatichydrocarbon radical containing at least one carbon-carbon double bondand having the number of carbon atoms designated, e.g. C₂-C₄alkenylmeans an alkenyl radical having from 2 to 4 carbon atoms; C₂-C₆alkenylmeans an alkenyl radical having from 2 to 6 carbon atoms. Non-limitingalkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl,n-pentenyl and hexenyl. An alkenyl group may be unsubstituted orsubstituted by one or more substituents which may be the same ordifferent, each substituent being independently selected from the groupconsisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy,alkoxy, —O-aryl, alkoxyalkyl, alkylthio, —NH₂, —NH(C_(m)-C_(n)alkyl),—N(C_(m)-C_(n)alkyl)₂, —NH(C₃-C_(n)cycloalkyl), —OC(O)C_(m)-C_(n)alkyl,—O—C(O)aryl, —O—C(O)C₃-C_(n)cycloalkyl, —C(O)OH and—C(O)OC_(m)-C_(n)alkyl. It is generally preferred that the alkenyl groupis unsubstituted, unless otherwise indicated.

“C₂-C_(n)alkynyl” represents a straight or branched aliphatichydrocarbon radical containing at least one carbon-carbon triple bondand having the number of carbon atoms designated, e.g. C₂-C₄alkynylmeans an alkynyl radical having from 2 to 4 carbon atoms; C₂-C₆alkynylmeans an alkynyl radical having from 2 to 6 carbon atoms. Non-limitingalkenyl groups include ethynyl, propynyl, 2-butynyl and 3-methylbutynylpentynyl and hexynyl. An alkynyl group may be unsubstituted orsubstituted by one or more substituents which may be the same ordifferent, each substituent being independently selected from the groupconsisting of halo, C₂-C_(n)alkenyl, C₂-C_(n)alkynyl, aryl,C₃-C_(n)cycloalkyl, cyano, hydroxy, C_(m)-C_(n)alkoxy, —O-aryl,—C_(m)-C_(n)alkoxyC_(m)-C_(n)alkyl, C_(m)-C_(n)alkylthio, —NH₂,—NH(C_(m)-C_(n)alkyl), —N(C_(m)-C_(n)alkyl)₂, —NH(C₃-C_(n)cycloalkyl),—O—C(O)—C_(m)-C_(n)alkyl, —O—C(O)aryl, —OC(O) C₃-C_(n)cycloalkyl,—C(O)OH and —C(O)OC_(m)-C_(n)alkyl. It is generally preferred that thealkynyl group is unsubstituted, unless otherwise indicated.

The term “C_(m)-C_(n)haloalkyl” as used herein representsC_(m)-C_(n)alkyl wherein at least one C atom is substituted with ahalogen (e.g. the C_(m)-C_(n)haloalkyl group may contain one to threehalogen atoms), preferably chloro or fluoro. Typical haloalkyl groupsare C₁-C₂haloalkyl, in which halo suitably represents fluoro. Exemplaryhaloalkyl groups include fluoromethyl, difluoromethyl andtrifluoromethyl.

The term “C_(m)-C_(n)hydroxyalkyl” as used herein representsC_(m)-C_(n)alkyl wherein at least one C atom is substituted with onehydroxy group. Typical C_(m)-C_(n)hydroxyalkyl groups areC_(m)-C_(n)alkyl wherein one C atom is substituted with one hydroxygroup. Exemplary hydroxyalkyl groups include hydroxymethyl andhydroxyethyl.

The term “Me” means methyl, and “MeO” means methoxy.

The term “C_(m)-C_(n)alkylcarbonyl” represents a radical of the formulaC_(m)-C_(n)alkylC(═O)— wherein the C_(m)-C_(n)alkyl moiety is as definedabove. Typically, “C_(m)-C_(n)alkylcarbonyl” is C₁-C₆alkylC(═O)—.

“C_(m)-C_(n)alkoxy” represents a radical C_(m)-C_(n)alkyl-O— whereinC_(m)-C_(n)alkyl is as defined above. Of particular interest isC₁-C₄alkoxy which includes methoxy, ethoxy, n-propoxy, isopropoxy,tert-butoxy, n-butoxy and isobutoxy. Methoxy and isopropoxy aretypically preferred. C₁-C₆alkoxy has a corresponding meaning, expandedto include all straight and branched chain isomers of pentoxy andhexoxy.

The term “halo” represents a halogen radical such as fluoro, chloro,bromo or iodo. Typically, halo groups are chloro and especially fluoro.

The term “C₃-C_(n)cycloalkyl” represents a cyclic monovalent alkylradical having the number of carbon atoms indicated, e.g.C₃-C₇cycloalkyl means a cyclic monovalent alkyl radical having from 3 to7 carbon atoms. Preferred cycloalkyl radicals for use in the presentinvention are C₃-C₄alkyl i.e. cyclopropyl and cyclobutyl. A cycloalkylgroup may be unsubstituted or substituted by one or more substituentswhich may be the same or different, each substituent being independentlyselected from the group consisting of halo, C₂-C_(n)alkenyl,C₂-C_(n)alkynyl, aryl, cycloalkyl, cyano, hydroxy, C_(m)-C_(n)alkoxy,aryloxy, C_(m)-C_(n)alkoxyC_(m)-C_(n)alkyl, C_(m)-C_(n)alkylthio, —NH₂,—NH(C_(m)-C_(n)alkyl), —N(C_(m)-C_(n)alkyl)₂, —NH(cycloalkyl),—O—C(O)C_(m)-C_(n)alkyl, —O—C(O)aryl, —O—C(O)C₃-C_(n)cycloalkyl, —C(O)OHand —C(O)Oalkyl. It is generally preferred that the cycloalkyl group isunsubstituted, unless otherwise indicated.

The term “C₃-C_(n)cycloalkenyl” represents a cyclic monovalent alkenylradical having the number of carbon atoms indicated, e.g.C₃-C₇cycloalkenyl means a cyclic monovalent alkyl radical having from 3to 7 carbon atoms, and including at least one unsaturated bond,typically one such bond. Preferred cycloalkenyl radicals for use in thepresent invention are C₅-C₆alkyl i.e. cyclopropenyl and cyclohexenyl. Acycloalkyl group may be unsubstituted or substituted by one or moresubstituents which may be the same or different, each substituent beingindependently selected from the group consisting of halo, alkenyl,alkynyl, aryl, cycloalkyl, cyano, hydroxy, alkoxy, aryloxy, alkoxyalkyl,alkylthio, —NH₂, —NH(C_(m)-C_(n)alkyl), —N(C_(m)-C_(n)alkyl)₂,—NH(C₃-C_(n)cycloalkyl), —O—C(O)C_(m)-C_(n)alkyl, —O—C(O)aryl,—O—C(O)C₃-C_(n)cycloalkyl, —C(O)OH and —C(O)OC_(m)-C_(n)alkyl whereinC_(m)-C_(n)alkyl, C₃-C_(n)cycloalkyl and aryl are as defined above. Itis generally preferred that the cycloalkyl group is unsubstituted,unless otherwise indicated.

The term “amino” represents NH₂.

Compounds of the invention can be prepared by the methods depicted inthe schemes below and as detailed in the experimental descriptions.Starting materials and reagents used in the preparation of the compoundsof the invention are available from commercial suppliers such as AldrichChemical Co., Bachem or Sigma, or they are prepared by methods known inthe art following literature procedures. The schemes below are merelyillustrative of some methods by which the compounds of the invention canbe prepared, and various modifications to these schemes can be made andwill be obvious to one skilled in the art.

A general route to compounds of the invention wherein R³ is a bond withthe adjacent carbon atom, thus providing a keto group, and R⁴ is H isillustrated in Scheme 1.

Reaction of methyl acetoacetate with an aldehyde R¹CHO under basicconditions such as in the presence of sodium hydride and butyl lithiumor similar provides the δ-hydroxy-β-keto ester (1a). The reaction istypically performed under anhydrous conditions in a solvent like THF orsimilar at a temperature below 5° C., such as between 0 and 5° C.Cyclisation of the afforded hydroxy compound effected by treatment witha base such as sodium hydroxide or similar followed by acidic treatmentprovides the pyran derivative (1 b). The O-substituent is thenintroduced by reaction with a desired acyl chloride R²COCl in thepresence of a base such as DBU or similar, thus providing the esterderivative (1c). Treatment finally with a base like pyrrolidinopyridineor similar at an elevated temperature, such as between 100° C. and 150°C., preferably around 130° C., effects the rearrangement and providesthe desired compound of formula I (1d).

Compounds of the invention wherein R³ is a bond with the adjacent carbonatom, thus providing a keto group, and R⁴ is optionally substitutedC₁-C₆alkyl, C₃-C₆cycloalkyl or C₃-C₆cycloalkylC₁-C₃alkyl are obtained byalkylation of the hydroxy group of compound 1d whereas compounds whereinR⁴ is an ester or an amino acid ester, i.e. C(O)R⁵ or C(O)CHR⁷NH₂respectively are achieved by acylation of the hydroxy group. Thesemethods are generally illustrated in Scheme 2.

Alkylation of the hydroxy group of compound 1d effected by reaction withan alkylating agent like an dialkyl sulphate, an alkyl sulfonate such asa triflate, tosylate or mesylate or an alkyl halide of the desired alkylgroup in the presence of a base such as sodium hydride or potassiumcarbonate or similar provides the alkylated derivative 2a.Alternatively, the group R^(4′) may be introduced by reaction with theappropriate alcohol R^(4′)—OH in the presence of an acid like p-toluenesulfonic acid or equivalent typically at elevated temperature, or byusing Mitsunobu conditions, i.e. in the presence of an azodicarboxylatesuch as DIAD or equivalent and triphenylphosphine.

Compounds of the invention wherein OR⁴ is an ester group (2b), i.e. R⁴is a group of formula C(O)R⁵ are obtained by acylation of alcohol 1ewith an acylating agent such as an alkyl anhydride in the presence ofpyridine, or an acid chloride or the like, whereas compounds wherein OR⁴is an amino acid ester group (2c), i.e. R⁴ is a group of formulaC(O)CHR⁷ are obtained by reaction of the alcohol (1d) with an aminoacid, optionally N-protected with a suitable N-protecting group such asa Boc, CBz or Fmoc group, in the presence of a suitable peptide couplingreagent such as EDAC or the like.

Compounds of the invention wherein R³ is H are obtained by reduction ofthe exocyclic keto function using reduction conditions such sodiumborohydride in the presence of cerium chloride in a solvent like THF orMeOH or similar, as illustrated in Scheme 3.

A route to compounds of the invention wherein R³ is optionallysubstituted C₁-C₆alkyl, C₃-C₆cycloalkyl or C₃-C₆cycloalkylC₁-C₃alkyl areobtained by alkylation of the hydroxy group of compound 3 whereascompounds wherein R³ is an ester or an amino acid ester, i.e. C(O)R⁵ orC(O)CHR⁷NH₂ respectively are obtained by acylation of the hydroxy group.These routes are depicted in Scheme 4.

Alkylation of the hydroxy group of compound 3 effected by reaction withan alkylating agent like an dialkyl sulphate, an alkyl sulfonate such asa triflate, tosylate or mesylate or an alkyl halide of the desired alkylgroup in the presence of a base such as sodium hydride or potassiumcarbonate or similar provides the alkylated derivative 4a.Alternatively, the group R^(3′) may be introduced by reaction with theappropriate alcohol R^(3′)—OH in the presence of an acid like p-toluenesulfonic acid or equivalent typically at elevated temperature. Compoundsof the invention wherein OR³ is an ester group, i.e. R³ is a group offormula C(O)R⁵ are obtained by acylation of alcohol 3 with an acylatingagent such as an alkyl anhydride in the presence of pyridine, or an acidchloride or the like, whereas compounds wherein OR³ is an amino acidester group (4c), i.e. R³ is a group of formula C(O)CHR⁷NH₂ are obtainedby reaction of the alcohol 3 with an amino acid, which is optionallyN-protected with a suitable N-protecting group such as a Boc, CBz orFmoc group, in the presence of a suitable peptide coupling reagent suchas EDAC or the like.

To obtain compounds of formula I wherein R⁴ is H, and R³ together withthe oxygen atom to which it is attached form an ether or acyloxy moiety,a protecting strategy as illustrated in Scheme 5 can be used.

Protection of the hydroxy group of compound 1d as for instance an ethersuch as a THP ether or a trityl ether or the like or as a silyl ethersuch as tert.butyl dimethylsilyl or tert.butyl diphenylsilyl or similarprovides protected derivative 5a. Reduction of the keto group to analcohol can then be effected using e.g. sodium borohydride in thepresence of cerium chloride in a solvent like THF or MeOH or similarconditions provide the exocyclic alcohol 5b. Alkylation or acylationusing the desired alkylating or acylating agent respectively usingconditions as described above followed by removal of the protectinggroup(s), then provides the O-substituted compounds 5c, 5d and 5e.

The pharmaceutical or veterinary compositions of the invention may beformulated in conventional manner, together with other pharmaceuticallyacceptable excipients if desired, into forms suitable for oral,parenteral, or topical administration. The modes of administration mayinclude parenteral, for example, intramuscular, subcutaneous andintravenous administration, oral administration, topical administrationand direct administration to sites of infection such as intraocular,intraaural, intrauterine, intranasal, intramammary, intraperitoneal,intralesional, etc.

The pharmaceutical or veterinary compositions of the invention may beformulated for oral administration. Traditional inactive ingredients maybe added to provide desirable colour, taste, stability, bufferingcapacity, dispersion, or other known desirable features. Examplesinclude red iron oxide, silica gel, sodium laurel sulphate, titaniumdioxide, edible white ink, and the like. Conventional diluents may beused to make compressed tablets. Both tablets and capsules may bemanufactured as sustained-release compositions for the continual releaseof medication over a period of time. Compressed tablets may be in theform of sugar coated or film coated tablets, or enteric-coated tabletsfor selective disintegration in the gastrointestinal tract. Liquiddosage forms for oral administration may contain colouring and/orflavouring to increase patient compliance. As an example, the oralformulation comprising compounds of the invention may be a tabletcomprising any one, or a combination of, the following excipients:calcium hydrogen phosphate dehydrate, microcrystalline cellulose,lactose, hydroxypropyl methyl cellulose, and talc.

The compositions described herein may be in the form of a liquidformulation. Examples of preferred liquid compositions includesolutions, emulsions, injection solutions, solutions contained incapsules. The liquid formulation may comprise a solution that includes atherapeutic agent dissolved in a solvent. Generally, any solvent thathas the desired effect may be used in which the therapeutic agentdissolves and which can be administered to a subject. Generally, anyconcentration of therapeutic agent that has the desired effect can beused. The formulation in some variations is a solution which isunsaturated, a saturated or a supersaturated solution. The solvent maybe a pure solvent or may be a mixture of liquid solvent components. Insome variations the solution formed is an in situ gelling formulation.Solvents and types of solutions that may be used are well known to thoseversed in such drug delivery technologies.

The composition described herein may be in the form of a liquidsuspension. The liquid suspensions may be prepared according to standardprocedures known in the art. Examples of liquid suspensions includemicro-emulsions, the formation of complexing compounds, and stabilisingsuspensions. The liquid suspension may be in undiluted or concentratedform. Liquid suspensions for oral use may contain suitablepreservatives, antioxidants, and other excipients known in the artfunctioning as one or more of dispersion agents, suspending agents,thickening agents, emulsifying agents, wetting agents, solubilisingagents, stabilising agents, flavouring and sweetening agents, colouringagents, and the like. The liquid suspension may contain glycerol andwater.

The composition described herein may be in the form of an oral paste.The oral paste may be prepared according to standard procedures known inthe art.

The composition described herein may be in the form of a liquidformulation for injection, such as intra-muscular injection, andprepared using methods known in the art. For example, the liquidformulation may contain polyvinylpyrrolidone K30 and water.

The composition described herein may be in the form of topicalpreparations. The topical preparation may be in the form of a lotion orcream, prepared using methods known in the art. For example, a lotionmay be formulated with an aqueous or oily base and may include one ormore excipients known in the art, functioning as viscosity enhancers,emulsifying agents, fragrances or perfumes, preservative agents,chelating agents, pH modifiers, antioxidants, and the like. For example,the topical formulation comprising one or more compounds of theinvention may be a gel comprising anyone, or a combination of, thefollowing excipients: PEG 8000, PEG 4000, PEG 200, glycerol, propyleneglycol. The compound of formula I may further be formulated into a soliddispersion using SoluPlus (BASF, http://www.soluplus.com) and formulatedwith any one, or a combination of, the following excipients: PEG 8000,PEG 4000, PEG 200, glycerol, and propylene glycol.

For aerosol administration, the composition of the invention is providedin a finely divided form together with a non-toxic surfactant and apropellant. The surfactant is preferably soluble in the propellant. Suchsurfactants may include esters or partial esters of fatty acids.

The compositions of the invention may alternatively be formulated fordelivery by injection. As an example, the compound is delivered byinjection by any one of the following routes: intravenous,intramuscular, intradermal, intraperitoneal, and subcutaneous.

The compositions of the invention may alternatively be formulated usingnanotechnology drug delivery techniques such as those known in the art.Nanotechnology-based drug delivery systems have the advantage ofimproving bioavailability, patient compliance and reducing side effects.

The formulation of the composition of the invention can include thepreparation of nanoparticles in the form of nanosuspensions ornanoemulsions, based on compound solubility. Nanosuspensions aredispersions of nanosized drug particles prepared by bottom-up ortop-down technology and stabilised with suitable excipients. Thisapproach may be applied to the compounds of the invention which can havepoor aqueous and lipid solubility, in order to enhance saturationsolubility and improve dissolution characteristics. An example of thistechnique is set out in Sharma and Garg (2010) (Pure drug andpolymer-based nanotechnologies for the improved solubility, stability,bioavailability, and targeting of anti-HIV drugs. Advanced Drug DeliveryReviews, 62: p. 491-502). Saturation solubility will be understood to bea compound-specific constant that depends on temperature, properties ofthe dissolution medium, and particle size (<1-2μιτι).

The composition of the invention may be provided in the form of ananosuspension. For nanosuspensions, the increase in the surface areamay lead to an increase in saturation solubility. Nanosuspensions arecolloidal drug delivery systems, consisting of particles below 1μιη.Compositions of the invention may be in the form of nanosuspensionsincluding nanocrystalline suspensions, solid lipid nanoparticles (SLNs),polymeric nanoparticles, nanocapsules, polymeric micelles anddendrimers. Nanosuspensions may be prepared using a top-down approachwhere larger particles may be reduced to nanometer dimensions by avariety of techniques known in the art including wet-milling andhigh-pressure homogenisation. Alternatively, nanosuspensions may beprepared using a bottom-up technique where controlled precipitation ofparticles may be carried out from solution.

The composition of the invention may be provided in the form of ananoemulsion. Nanoemulsions are typically clear oil-in-water orwater-in-oil biphasic systems, with a droplet size in the range of100-500 nm, and with compounds of interest present in the hydrophobicphase. The preparation of nanoemulsions may improve the solubility ofthe compounds of the invention described herein, leading to betterbioavailability. Nanosized suspensions may include agents forelectrostatic or steric stabilisation such as polymers and surfactants.Compositions in the form of SLNs may comprise biodegradable lipids suchas triglycerides, steroids, waxes and emulsifiers such as soybeanlecithin, egg lecithin, and poloxamers. The preparation of a SLNpreparation may involve dissolving/dispersing drug in melted lipidfollowed by hot or cold homogenisation. If hot homogenisation is used,the melted lipidic phase may be dispersed in an aqueous phase and anemulsion prepared. This may be solidified by cooling to achieve SLNs. Ifcold homogenisation is used, the lipidic phase may be solidified inliquid nitrogen and ground to micron size. The resulting powder may besubjected to high-pressure homogenisation in an aqueous surfactantsolution.

The Compounds of Formula I as described herein may be dissolved inoils/liquid lipids and stabilised into an emulsion formulation.Nanoemulsions may be prepared using highland low-energy dropletreduction techniques. High-energy methods may include high-pressurehomogenisation, ultrasonication and microfluidisation. If the low-energymethod is used, solvent diffusion and phase inversion will generate aspontaneous nanoemulsion. Lipids used in nanoemulsions may be selectedfrom the group comprising triglycerides, soybean oil, safflower oil, andsesame oil. Other components such as emulsifiers, antioxidants, pHmodifiers and preservatives may also be added.

The composition may be in the form of a controlled-release formulationand may include a degradable or non-degradable polymer, hydrogel,organogel, or other physical construct that modifies the release of thecompound. It is understood that such formulations may include additionalinactive ingredients that are added to provide desirable colour,stability, buffering capacity, dispersion, or other known desirablefeatures. Such formulations may further include liposomes, such asemulsions, foams, micelles, insoluble monolayers, liquid crystals,phospholipid dispersions, lamellar layers and the like. Liposomes foruse in the invention may be formed from standard vesicle-forming lipids,generally including neutral and negatively charged phospholipids and asterol, such as cholesterol. 100205] The formulations of the inventionmay have the advantage of increased solubility and/or stability of thecompounds, particularly for those formulations prepared usingnanotechnology techniques. Such increased stability and/or stability ofthe compounds of Formula I may improve bioavailability and enhance drugexposure for oral and/or parenteral dosage forms. Throughout thisspecification, unless the context requires otherwise, the word“comprise” or variations such as “comprises” or “comprising”, will beunderstood to imply the inclusion of a stated integer or group ofintegers but not the exclusion of any other integer or group ofintegers.

In addition to the definitions above, the following abbreviations areused in the examples and synthetic schemes below. If an abbreviationused herein is not defined, it has its generally accepted meaning.

DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene

DCM Dichloromethane

DMF N,N-Dimethylformamide

ES Electrospray

Et₃N Triethylamine

EtOAc Ethyl acetate

EtOH Ethanol

LC Liquid chromatography

HOAc Acetic acid

HPLC High performance liquid chromatography

MeCN Acetonitrile

MeOH Methanol

MIC Minimum inhibition concentration

MS Mass spectrometry

PBS Phosphate buffered saline

Pg Protecting group

Ph Phenyl

THF Tetrahydrofuran

TFA Trifluoroacetic acid

VPB Vegetable Peptone Broth

SYNTHESIS EXAMPLES Example 1

Step a) Methyl 5-hydroxy-3-oxotetradecanoate (1a)

Fresh NaH (60% dispersion in mineral oil, 0.88 g, 22 mmol) was addedunder nitrogen to THF (50 ml) and the suspension was cooled to 0° C.Methyl acetoacetate (2.16 ml, 20 mmol) was added drop wise while keepingthe inside temp below 5° C. The stirring was continued at 0° C. for anadditional 10 min, then 2.5 M n-BuLi (8.4 ml, 21 mmol) was added dropwise while keeping the inside temperature below 5° C. The stirring wascontinued for an additional 10 min, then, decanal (3.67 ml, 20 mmol) wasadded and the reaction mixture was stirred for an additional 30 minallowing the temperature to slowly increase to about 10° C. The reactionwas quenched, with 6M HCl until acidic followed by addition of water andether. The water phase was extracted twice with ether and the combinedorganic phases were carefully washed with brine to neutral pH and dried(Na₂SO₄). After filtration, the organic solvent was evaporated and acrude product (5.41 g) was obtained. The crude was recrystallized fromn-hexanes and after standing at 5° C. a first crop of product (1.6 g)was obtained by transferring the mother liquid with a thin pipette at °0C. A second crop (1.29 g) was obtained giving a total yield of 2.9 g(53%).

Step b) 6-Nonyldihydro-2H-pyran-2,4(3H)-dione (1b)

The compound from step a (1a, 1.61 g, 5.9 mmol) was mixed with 1M NaOH(15 ml). The reaction mixture was stirred at 0° C. for about 1 h, then12M HCl (˜3 ml) was added and the reaction mixture was allowed to attainroom temperature overnight. Water (15 ml) was added and the mixture wasextracted 3 times with DCM. The combined organic phases were washed withbrine, dried (Na₂SO₄) and concentrated which gave crude title compound(1.26 g, 89%). The afforded crude product was taken to the next stepwithout further purification.

Step c) 2-Nonyl-6-oxo-3,6-dihydro-2H-pyran-4-yl butyrate (1c)

The compound from step b (1b, 1.26 g, 5.24 mmol) was dissolved intoluene (15 ml) and DBU (1.18 ml, 7.86 mmol) was added. The reactionsolution was cooled to 0° C. and butyryl chloride (0.82 ml, 7.86 mmol)was added drop wise. The reaction mixture was stirred at 0° C. for 1 hwhere after the temperature was allowed to reach room temperature. After3 hours, water was added and the reaction mixture was extracted twicewith ether. The combined organic phases were washed with HCl solution,NaHCO₃ solution and brine. The organic phase was dried (Na₂SO₄) andconcentrated. The afforded crude product was purified on a silica gelcolumn eluted with hexanes/ether 3:1 which gave the title compound (1.18g, 72.5%). LC-MS ES+311.07 [M+H]⁺.

¹H NMR (400 MHz, CDCl₃) δ 7.25, 5.90, 5.89, 4.50, 4.49, 4.48, 4.47,4.47, 4.46, 4.45, 4.44, 4.44, 4.43, 2.68, 2.68, 2.66, 2.65, 2.64, 2.64,2.61, 2.61, 2.47, 2.45, 2.44, 2.43, 2.41, 2.40, 2.39, 2.35, 2.33, 2.31,2.26, 2.24, 2.23, 2.22, 2.15, 1.86, 1.85, 1.84, 1.83, 1.83, 1.82, 1.81,1.80, 1.80, 1.79, 1.79, 1.78, 1.77, 1.75, 1.73, 1.72, 1.70, 1.68, 1.66,1.65, 1.64, 1.63, 1.63, 1.61, 1.60, 1.54, 1.52, 1.51, 1.49, 1.48, 1.48,1.42, 1.41, 1.39, 1.38, 1.29, 1.28, 1.25, 1.01, 0.99, 0.97, 0.95, 0.93,0.91, 0.89, 0.87, 0.86, 0.06, −0.01.

Step d) 3-Butyryl-6-nonyl-4-hydroxy-5,6-dihydro-2H-pyran-2-one (1d)

The compound from step c (1c, 2.5 g, 8.05 mmol) was dissolved in toluene(50 ml) and 4-pyrrolidinopyridine (60 mg, 0.4 mmol) was added. Thereaction was heated at 130° C. for 4 hrs. The solvent was evaporated andthe residue purified on a silica gel column eluted with ether/hexanes1:5 which gave a crude product as a solid (1.89 g). The solid wasrecrystallized from 10 ml warm hexanes and stored at 5° C. for 3 days.The mother liquid was removed by a thin pipette at 0° C. The crystalswere then washed with 1 ml cold hexane. The white crystals were dried invacuum which gave the title compound (1.03 g, 41%). The purity wasdetermined to 99% by LC/UV analysis. LC-MS ES+311.2 [M+H]⁺.

¹H NMR (400 MHz, CDCl₃) δ 17.88, 16.18, 7.25, 4.47, 4.45, 4.43, 4.37,4.36, 4.36, 4.36, 4.35, 4.35, 4.34, 4.34, 4.33, 4.33, 4.32, 4.32, 4.32,4.31, 3.08, 3.06, 3.06, 3.04, 3.04, 3.03, 3.02, 3.02, 3.00, 2.99, 2.98,2.98, 2.97, 2.97, 2.96, 2.94, 2.93, 2.92, 2.69, 2.66, 2.65, 2.62, 2.61,2.60, 2.57, 2.56, 2.56, 2.55, 2.55, 2.23, 1.83, 1.81, 1.81, 1.80, 1.80,1.79, 1.79, 1.78, 1.78, 1.77, 1.77, 1.77, 1.76, 1.76, 1.75, 1.74, 1.74,1.72, 1.71, 1.71, 1.69, 1.69, 1.69, 1.67, 1.67, 1.65, 1.65, 1.64, 1.63,1.63, 1.62, 1.62, 1.61, 1.59, 1.58, 1.56, 1.53, 1.51, 1.50, 1.50, 1.48,1.48, 1.47, 1.47, 1.46, 1.45, 1.44, 1.42, 1.41, 1.39, 1.38, 1.37, 1.32,1.31, 1.28, 1.25, 1.02, 1.00, 0.98, 0.96, 0.88, 0.87, 0.85, −0.02.

Example 2A & 2B Separation of the Enantiomers of Compound 1d

Compound 1d (100 mg) was resolved by chiral chromatography using a LuxCell-3 column eluted with A=hexane, B=80% hexane, 20% EtOH+1% TFA. Theenantiomeric purity was determined by an analytical Lux Cell-3 columnand the absolute configuration was postulated based on relatedPodoblastins cf. J. Pesticide Sci. 13, 605-613 (1988).

Peak 1—Compound 2A:

Amount 44 mg, enantiomeric purity 99.9%, [α]_(D)−31 (c=0.245, DCM),absolute configuration (6R)

Peak 2—Compound 2B:

Amount 38 mg, enantiomeric purity 99.8%, [α]D+33° (c=0.24, DCM),absolute configuration (6S).

Example 29, Acylation of Compound 1d

5-Butyryl-2-nonyl-6-oxo-3,6-dihydro-2H-pyran-4-yl propionate (29)

Propionyl chloride (0.013 mL, 0.150 mmol) was added at 0° C. to asolution of compound 1d (31.0 mg, 0.100 mmol) in ACN (3.00 mL) and TEA(0.021 mL, 0.150 mmol). The reaction mixture was left at roomtemperature and stirred for 18 h. DCM was added and the solution waswashed with water. The organic layer was dried (Na₂SO₄), filtered andconcentrated, which gave the title compound.

Compound 3 to 28 listed in Table 1 below were prepared according to themethod described in Example 1. ¹H NMR was ran for the penultimate andlast step for Compounds 2-28 and the spectra were found to be inaccordance with the spectra for compounds 1c and 1d.

BIOLOGICAL EXAMPLES Biological Example 1—Determination of MinimumInhibitory Concentration (MIC) Against Gram-Positive and Gram-NegativePathogens

At first (Table 1), MIC is determined by broth micro-dilution method.The test medium is Antibiotic Medium 3 (AM3; Difco Ltd) mixed withPhosphate Buffered Saline (PBS) in proportions 1:1. Selected MIC valuesare verified using cation-adjusted Mueller Hinton Broth (MHB; BD). Cellconcentration of tested pathogen is adjusted to between 3 and 5×10⁵cells per ml. To verify cell concentration of tested pathogen, viablecell counts are performed directly after each MIC-test by dilutionmethod with PBS as a dilution solvent. Cells are counted on the agarplates with 50% strength Vegetable Peptone Broth (50% VPB; Oxoid Ltd)after 16 to 20 h of incubation at 37° C.

In order to establish MIC, appropriate volumes of solutions of testcompounds (100, 10, 1 and if needed 0.1 μg/ml) dissolved in MeOH aredispensed into wells of 96-wells microtiter plates and the solvent isevaporated. MIC of the test compounds is detected in a concentrationrange between 0.05 and 20 μg per ml. Cells of pathogen suspended in testmedium are distributed to the wells and the plates are incubated for 16to 20 h at 37° C. Negative controls are wells with the test medium onlyand positive controls are wells with the pathogen in test medium. TheMIC is defined as the lowest concentration of each compound with novisible growth of pathogen.

Example compounds of formula I were examined for antibacterial activityagainst the Gram-positive bacterium Staphylococcus aureus strainLMG15975 using the above described method (Table 1).

MIC of Example 1 and its selected derivatives (Ex. 14, Ex. 16 and Ex.17) (Table 5) is, additionally, determined using the brothmicro-dilution method according to M07-A9 Clinical and LaboratoryStandards Institute (CLSI) guidelines. All pathogens are testedaccording to M07-A9 and M26 CLSI guidelines using the appropriate brothmedium. Negative controls (lack of bacterial cells) and growth controlare included in the plates. S. aureus ATCC 29213 and S. pneumoniaeATCC49619 are evaluated as assay quality control strain. The MIC isdefined as the lowest concentration of each compound with no visiblegrowth of pathogen. The MIC of Example 1 is compared to the MIC ofciprofloxacin (Table 6).

Antibacterial activity of the tested compounds determined as MIC issummarized in Tables 1-7.

TABLE 1

Compound R¹ R² MS [M + H]⁺ MIC (μg/ml) MIC (μM) CC₅₀ (μg/mL) Ex. 1 nonylPropyl 311.2 0.8 2.5 33 Ex. 2A nonyl Propyl 311.2 1-2 3.2-6.4 27 Ex. 2Bnonyl Propyl 311.2 0.4-0.6 1.3-1.9 25 Ex. 3 decyl Butyl 339.2 0.4-0.61.2-1.8 13 Ex. 4 1-trans-nonenyl Propyl 309.33 0.8-1.2 2.6-3.9 36 Ex. 5nonyl Ethyl 297.4 0.65 2.2 50 Ex. 6 octyl Ethyl 283.3 1.2 4.3 55 Ex. 7octyl methyl 269.3 2.5 9.3 65 Ex. 8 nonyl Butyl 325.4 ≤0.6 ≤1.9 36 Ex. 9nonyl methyl 283.3 1.5-2.5 2.1-4.3 71 Ex. 10 decyl Ethyl 311.4 0.4-0.81.3-2.6 60 Ex. 11 decyl methyl 297.2 0.8-1.2 2.7-4.0 40 Ex. 12 decylPropyl 325.2 0.6-0.8 1.9-2.6 25 Ex. 13 octyl propyl 297.2 0.8-1.52.7-5.1 30 Ex. 14 nonyl cyclohexyl- 365.3 0.25 ≤0.3 14 methyl Ex. 15nonyl isobutyl 325.2 0.2-0.4 0.6-1.2 21 Ex. 16 undecyl propyl 339.20.1-0.2 0.3-0.6 19 Ex. 17 undecyl butyl 353.2 0.1-0.2 0.3-0.6 12 Ex. 18nonyl pentyl 339.3 0.6-0.8 1.8-2.4 15 Ex. 19 7-cyanoheptyl propyl 308.140-50 133-166 >100 Ex. 20 7-methoxy- propyl 313.1 20-30 64-96 80 heptylEx. 21 2,6-dimethyl- propyl 309.2 2-3 6.5-9.7 na hept-5-enyl Ex. 22cyclopentyl- propyl 267.2  6-12 22-45 na methyl Ex. 23 6-methoxy-cyclopentyl- 339.2 10-20 29-59 na hexyl methyl Ex. 24 nonyl 2-methoxy-327.2 2-5 6.1-15  na ethyl Ex. 25 6-methoxy- cyclopropyl- 311.2 >20 >64na hexyl methyl Ex. 26 nonyl (tetrahydro- 367.2 0.5-1   1.4-2.7 na2H-pyran-4- yl)methyl Ex. 27 7-cyanoheptyl (tetrahydro- 364.2 2-55.5-14  na 2H-pyran-4- yl)methyl Ex. 28 7-cyanoheptyl cyclohexyl-362.2 >20 >55 na methyl Test comp. ethyl propyl 213.0 >50 >240 >100 na =not available

TABLE 2 Activity for selected compounds, with increasing lengths of R₁or R₂, towards A. baumannii (G−) and S. aureus (G+).

MIC (μg/ml) MIC (μg/ml) Compound R₁ R₂ A. baumannii S. aureus Ex. 9nonyl methyl >64 1.5-2.5 Ex. 5 nonyl ethyl 32-48 0.7 Ex. 1 nonyl propyl16-24 0.8 Ex. 8 nonyl butyl  8-16 <0.6   Ex. 18 nonyl pentyl 4-8 0.6-0.8Comp. Ex. ethyl propyl >64 >50     Nat. prod.* heptyl propyl >64 1-2 Ex.13 octyl propyl 24-32 0.8-1.5 Ex. 1 nonyl propyl  8-16 0.8 Ex. 12 decylpropyl  8-16 0.6-0.8 Ex. 16 undecyl propyl  <4 0.1-0.2 *The compound wasisolated from cultures of a soil bacterium.

TABLE 3 Sensitivity of 4 mupirocin-resistant strains of Staphylococcusaureus against selected example compounds (Table 1). All strains had MICvalues above 256 ug/ml for mupirocin. MIC (μg/ml) Isolate Ex. 1 Ex. 8Ex. 14 Ex. 24 S. aureus V55 ≤0.1 ≤0.1 ≤0.1 >1 ≤ 2 S. aureus V86 >0.1 ≤0.25 ≤0.1 ≤0.1 >1 ≤ 2 S. aureus V308 >0.1 ≤ 0.25 >0.1 ≤ 0.25 ≤0.1 >1 ≤ 2S. aureus V321 >0.1 ≤ 0.25 ≤0.1 ≤0.1 >1 ≤ 2

TABLE 4 Sensitivity of Staphylococcus aureus, Mycobacterium smegmatis,M. frederiksbergense and Bacillus cereus, against selected examplecompounds (Table 1) MIC (μg/ml) Compound S. aureus M. smegmatis M.frederiksbergense B. cereus Ex. 1  0.8 >0.5 ≤ 1   ≤0.5 ≤1 Ex. 5 0.7 >0.5 ≤ 1   >1 ≤ 2  ≤1 Ex. 8 ≤0.6 ≤4 ≤4   ≤1 Ex. 10 >0.4 ≤ 0.8 >1 ≤2 ≤0.5 ≤1 Ex. 14 ≤0.1 >1 ≤ 2 >0.5 ≤ 1  ≤1 Ex. 17 >0.1 ≤ 0.2 >1 ≤ 2 >0.5≤ 1  ≤1 Ex. 23 >10 ≤ 20 >4 ≤ 8  >8 ≤ 16 >4 ≤ 8

TABLE 5 Antimicrobial Susceptibility Test (AST) by Broth MicrodilutionMethod according to M07-A9 CLSI guidelines. The tested compounds areExample 14, 16, 17 and 1 (Table 1)

Main phenotype of MIC (μg/mL) Organism antibiotic resistance Ex. 14 Ex.16 Ex. 17 Ex. 1 S. aureus ATCC29213 QC strain 2 4 16 4 S. aureus MO17Methicillin-sensitive 1 4 4 2 S. aureus PICI Methicillin-resistant 1 4 84 S. aureus 7280 Methicillin-resistant 2 8 8 NT S. epidermis RO7Methicillin-sensitive 0.5 0.5 0.5 1 S. epidermis Staph 1Methicillin-resistant 0.5 0.5 0.5 NT S. epidermis TA12Methicillin-resistant 0.5 1 0.5 2 E. faecium 7 Vancomycin-resistant 1 21 2 E. faecalis 24 Vancomycin-sensitive 1 1 1 2 E. faecalis 14Vancomycin-resistant 1 1 1 NT S. aureus LMG 15975 (SLU)Methicillin-resistant 0.25 0.1-0.2 0.1-0.2 0.8 S. aureus 4384 (SSI)Methicillin-resistant NT NT NT 2 CC₅₀ (μg/mL) 14 19 12 38

TABLE 6 Antimicrobial Susceptibility Test (AST) by Broth MicrodilutionMethod according to M07-A9 CLSI guidelines. The tested compound isExample 1, R¹ = nonyl, R2 = propyl, in comparison to Ciprofloxacin MainPhenotype of Antibiotic MIC (μg/mL) Organism Resistance Ex 1Ciprofloxacin S. aureus ATCC 29213 QC strain 4 0.125 S. aureus 4216Tetracycline-resistant 2 >32 S. aureus 853E Macrolide-sensitive 2 0.25S. aureus 6 Holland Macrolide-resistant (M) 1 1 S. aureus 56 JapanMacrolide-resistant (MLSi) 4 >8 S. aureus PK2 Macrolide-resistant (MLSc)2 0.25 S. aureus MO 17 MSSA 2 0.5 S. aureus PICI MRSA 4 >32 S. aureus FR131 Quinolone-resistant 2 >32 S. pneumoniae ATCC 49619 QC strain 4 0.5S. pneumoniae 158bg Macrolide-resistant (M) 4 2 S. pneumoniae SP030Macrolide- sensitive 8 0.5 S. pneumoniae 956 Macrolide-resistant (MLSc)4 1 S. pneumoniae 19A-TN Quinolone-sensitive 8 0.5 S. pneumoniae TE122-024 Quinolone-resistant 8 2 S. pneumoniae TE 122-026Tetracycline-resistant 4 1 S. pneumoniae BAA1407 Macrolide-resistant(M + MLS) 8 1 S. pyogenes 21 Finland Macrolide-resistant (MLSc) 8 0.25S. pyogenes 2 Finland Macrolide-resistant (M) 8 5 S. pyogenes 29A-TFMacrolide- sensitive 8 0.25 S. epidermidis RO7 MSSE 1 0.125 S.epidermidis TA12 MRSE 2 >32 E. faecalis E.cocco 24 Vancomycin -sensitive2 0.5 E. faecalis E.cocco 12 Vancomycin -resistant 2 32 E. faecium E.coc7 Vancomycin -resistant 2 >32

TABLE 7 Table 7 shows results for Example 14) and Example 1 testedagainst a panel of Gram-negative bacteria. μg/mL EN no. Species Other IDEx. 14 Ex. 1 Wide range of Gram-negative species EN0454 Serratiamarescens G− PKL 3692 >64 >64 EN0455 Micrococcus luteus G+ PKL 3693 0.50.5 EN0457 Proteus mirabilis G− PKL 3695 >64 >64 EN0458 E. coli B G− PKL3698 >64 >64 EN0459 P. aeruginosa G− PKL 3699 >64 >64 EN0460 P.aeruginosa G− PKL 3704 >64 >64 EN0461 Enterobacter aerogenes G− PKL3706 >64 >64 EN0456 S. epidermidis G+ PKL 3694 1 1 EN0465 Citrobactersp. G− PKL 3710 >64 >64 EN0466 S. typhimurium G− TH6509 >64 >64 EN0180A. Pittii/nosocomialis G− >64 >64 EN0392 K. oxytoca G− >64 >64 EN0422 P.aeruginosa G− >64 >64 EN0445 P. mirabilis ST19 G− >64 >64 ENABLE PrimaryMIC panel strains EN001 E. coli G− WT >64 >64 EN002 E. coli G− DtolC >644 EN003 E. coli G− D22 (lps mut) >64 >64 EN004 P. aeruginosa G−WT >64 >64 EN005 P. aeruginosa G− Efflux mut >64 2 EN006 K. pneumoniaeG− WT >64 >64 EN007 A. baumanii G− WT >64 >64 EN008 S. aureus G+ Gram+ 22 ENABLE L2C MIC panel strains (isogenic efflux pairs) EN0010 K.pneumoniae G− >64 >64 EN0011 K. pneumoniae G− Efflux mut >64 8 EN0012 E.coli G− >64 >64 EN0013 E. coli G− Del-tolC 16 0.5 EN0014 P. aeruginosaG− >64 >64 EN0015 P. aeruginosa G− Efflux mut >64 >64 EN0016 A. baumaniiG− >64 >64 EN0017 A. baumanii G− Efflux mut >64 >64Cytotoxicity Assay

Cytotoxicity of the compounds was evaluated in HepG2 cells as follows:HepG2 (ATCC cat. no. HB-8065) hepatocellular carcinoma cells weremaintained in Dulbecco's Modified Eagle Medium supplemented with 10%heat inactivated fetal calf serum, penicillin (50 U/ml) and streptomycin(50 μg/ml). Briefly, cells were passaged into 96 well microplates (1×10⁴cells/well) and the next day, test compounds were added in two-foldserial dilutions starting from a final top concentration of 100 μM.After another 48 hours of incubation at 37°, the number of viable cellsin each well was assessed by using a water-soluble tetrazolium salt,WST-8 assay (cell counting Kit-8 from Dojindo). The concentrationcausing 50% decrease in cell viability (CC₅₀) was determined.

Biological Example 2, In Vivo Pharmacokinetics

Compound Example 1_(Table 1) was administered orally to triplicate micein a conventional 20% HPBCD vehicle. Two doses were administered, 10 and50 μmol/kg. Blood samples were taken at relevant timepoints over 48hours, as depicted in the Table 8 below, and the plasma concentration ofExample 1 determined by LC/MS/MS. The compound exhibited excellent dosedependent exposure. Macroscopic examination of the animals and theirbehaviour did not show any sign of adverse effect.

TABLE 8 Determination of Example 1 concentration in mouse plasmafollowing oral administration of Example 1 (50 μmol/kg) in a 20% HPBCDvehicle. Concentrations were determined at the indicated times usingLC/MS/MS. Concentration in plasma (μM) Time (h) Mouse 1 Mouse 2 Mouse 30.25 73 71 162 0.5 101 126 163 1 136 178 217 3 204 234 246 5 213 294 3207 266 240 280 24 169 152 94 48 17 11 15

Biological Example 3, In Vitro Absorption

Permeability

This experiment measures transport of inhibitors through the cells ofthe human gastroenteric canal. The assay uses the well-known Caco-2cells with a passage number between 40 and 60.

Apical to Basolateral Transport

Generally every compound will be tested in 2-4 wells. The basolateraland the apical wells will contain 1.5 mL and 0.4 mL transport buffer(TB), respectively, and the standard concentration of the testedsubstances is 10 μM. Furthermore all test solutions and buffers willcontain 1% DMSO. Prior to the experiment the transport plates arepre-coated with culture medium containing 10% serum for 30 minutes toavoid nonspecific binding to plastic material. After 21 to 28 days inculture on filter supports, the cells are ready for permeabilityexperiments.

Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denotedWash, row 2 “30 minutes” and row 3 “60 minutes”. Transport plate no 2comprises 3 rows of 4 wells, one denoted row 4 “90 minutes”, row 5 “120minutes and the remaining row unassigned.

The culture medium from the apical wells is removed and the inserts aretransferred to a wash row (No. 1) in a transport plate (plate no.1) outof 2 plates without inserts, which have already been prepared with 1.5mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A→Bscreening the TB in basolateral well also contains 1% Bovine SerumAlbumin.

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to theinserts and the cell monolayers equilibrated in the transport buffersystem for 30 minutes at 37° C. in a polymix shaker. After beingequilibrated to the buffer system the Transepithelial electricalresistance value (TEER) is measured in each well by an EVOM chop stickinstrument. The TEER values are usually between 400 to 1000Ω per well(depends on passage number used).

The transport buffer (TB, pH 6.5) is removed from the apical side andthe insert is transferred to the 30 minutes row (No. 2) and fresh 425 μLTB (pH 6.5), including the test substance is added to the apical (donor)well. The plates are incubated in a polymix shaker at 37° C. with a lowshaking velocity of approximately 150 to 300 rpm.

After 30 minutes incubation in row 2, the inserts are moved to newpre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60minutes), 4 (90 minutes) and 5 (120 minutes).

25 μL samples are taken from the apical solution after ˜2 minutes and atthe end of the experiment. These samples represent donor samples fromthe start and the end of the experiment.

300 μL will be taken from the basolateral (receiver) wells at eachscheduled time point and the post value of TEER is measured at the endthe experiment. To all collected samples acetonitrile will be added to afinal concentration of 50% in the samples. The collected samples will bestored at −20° C. until analysis by HPLC or LC-MS.

Basolateral to Apical Transport

Generally every compound will be tested in 2-4 wells. The basolateraland the apical wells will contain 1.55 mL and 0.4 mL TB, respectively,and the standard concentration of the tested substances is 10 μM.Furthermore all test solutions and buffers will contain 1% DMSO. Priorto the experiment the transport plates are precoated with culture mediumcontaining 10% serum for 30 minutes to avoid nonspecific binding toplastic material.

After 21 to 28 days in culture on filter supports the cells are readyfor permeability experiments. The culture medium from the apical wellsare removed and the inserts are transferred to a wash row (No. 1) in anew plate without inserts (Transport plate).

The transport plate comprises 3 rows of 4 wells. Row 1 is denoted “wash”and row 3 is the “experimental row”. The transport plate has previouslybeen prepared with 1.5 mL TB (pH 7.4) in wash row No. 1 and with 1.55 mLTB (pH 7.4), including the test substance, in experimental row No. 3(donor side).

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to theinserts in row No. 1 and the cell monolayers are equilibrated in thetransport buffer system for 30 minutes, 37° C. in a polymix shaker.After being equilibrated to the buffer system the TEER value is measuredin each well by an EVOM chop stick instrument.

The transport buffer (TB, pH 6.5) is removed from the apical side andthe insert is transferred to row 3 and 400 μL fresh TB, pH 6.5 is addedto the inserts. After 30 minutes 250 μL is withdrawn from the apical(receiver) well and replaced by fresh transport buffer. Thereafter 250μL samples will be withdrawn and replaced by fresh transport bufferevery 30 minutes until the end of the experiment at 120 minutes, andfinally a post value of TEER is measured at the end of the experiment. A25 μL samples will be taken from the basolateral (donor) compartmentafter ˜2 minutes and at the end of the experiment. These samplesrepresent donor samples from the start and the end of the experiment.

To all collected samples acetonitrile will be added to a finalconcentration of 50% in the samples. The collected samples will bestored at −20° C. until analysis by HPLC or LC-MS.

Calculation

Determination of the cumulative fraction absorbed, FA_(cum), versustime. FA_(cum) is calculated from:

${FA}_{cum} = {\sum\frac{C_{RI}}{C_{DI}}}$

Where C_(Ri) is the receiver concentration at the end of the interval iand C_(Di) is the donor concentration at the beginning of interval i. Alinear relationship should be obtained.

The determination of permeability coefficients (P_(app), cm/s) arecalculated from:

$P_{app} = \frac{\left( {k \cdot V_{R}} \right)}{\left( {A \cdot 60} \right)}$where k is the transport rate (min⁻¹) defined as the slope obtained bylinear regression of cumulative fraction absorbed (FA_(cum)) as afunction of time (min), V_(R) is the volume in the receiver chamber(mL), and A is the area of the filter (cm²).

TABLE 9 Reference compounds Category of absorption absorption in manMarkers in man (%) PASSIVE TRANSPORT Low (0-20%) Mannitol 16Methotrexate 20 Moderate (21-75%) Acyclovir 30 High (76-100%)Propranolol 90 Caffeine 100 ACTIVE TRANSPORT Amino acid transporterL-Phenylalanine 100 ACTIVE EFFLUX PGP-MDR1 Digoxin 30

Greater permeability through the gastrointestinal tissue is advantageousin that it allows for the use of a smaller dose to achieve similarlevels of exposure to a less permeable compound administered in a higherdose. A low dose is advantageous in that it minimizes the cost of goodsfor a daily dose, which is a crucial parameter in a drug which is takenfor protracted time periods.

The compound of Example 1 exhibited an excellent Caco2 permeabilityvalue of P_(app) 53 cmsec⁻¹.

The invention claimed is:
 1. A compound of Formula I:

wherein R¹ is substituted or unsubstituted C₂-C₂₀ alkenyl with 1-3unsaturated bonds; R² is selected from the group consisting ofsubstituted or unsubstituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, C₁-C₆ alkoxy C₁-C₃ alkyl, cyclopropylmethyl, cyclobutylmethyl,cyclopentylmethyl and cyclohexylmethyl; R³ is H or a bond along with theadjacent carbon defining a keto group, R⁵, C(O)R⁵, CR^(a)R^(b)OR⁵,CR^(a)R^(b)OC(O)R⁵, CR^(a)R^(b)OC(O)OR⁵ or C(O)CHR⁷NH₂; R⁴ is R⁵,C(O)R⁵, CR^(a)R^(b)OR⁵, CR^(a)R^(b)OC(O)R⁵, CR^(a)R^(b)OC(O)OR⁵ orC(O)CHR⁷NH₂; R⁵ and R⁶ are independently H, or selected from the groupconsisting of substituted or unsubstituted C₁-C₆alkyl, C₃-C₆cycloalkyland C₃-C₆cycloalkylC₁-C₃alkyl; R⁷ is H, C₁-C₆alkyl, C₃-C₆cycloalkyl andC₃-C₆cycloalkylC₁-C₃alkyl; wherein R^(a) and R^(b) are independentlyselected from H and methyl; or a pharmaceutically acceptable salt,stereoisomer, N-oxide or hydrate thereof; with the provisos that when R³is a bond, R⁴ is H, and R¹ and R² are both unsubstituted alkyl, then ifR¹ is C₆-alkyl, R² has at least two carbon atoms, and when R³ is a bond,R⁴ is H, and R¹ and R² are both unsubstituted C₁-C₂₀ alkyl, then R¹ hasat least 2 more carbon atoms than R².
 2. A compound according to claim1, wherein R¹ is C₂-C₂₀ alkenyl substituted with up to threesubstituents independently selected from the group consisting of halo,—OR⁵, —C(O)OR⁵, —C(O)R⁵, —OC(O)OR⁵, —NR⁵R⁶, —C(O)NR⁵R⁶, —OC(O)NR⁵R⁶,nitro, cyano and azido.
 3. A compound according to claim 1, which isracemic at the R¹ position.
 4. A compound according to claim 1, in whichR¹ is at least 75% enantiomerically enriched as regards one enantiomer,preferably greater than 90% enantiomerically enriched.
 5. A compoundaccording to claim 1, wherein R¹ is 1 trans-nonenyl; R² is selected fromethyl, propyl, butyl, methyl, cyclohexylmethyl, isobutyl and pentyl; R³is a bond along with the adjacent carbon defining a keto group; and R⁴is H.
 6. A compound according to claim 1, wherein in R², any alkyl,alkenyl, alkynyl, cycloalkyl or cycloalkenyl is substituted with up tothree substituents independently selected from the group consisting ofhalo, —OR⁵, —C(O)OR⁵, —C(O)R⁵, —OC(O)OR⁵, —NR⁵R⁶, —C(O)NR⁵R⁶,—OC(O)NR⁵R⁶, nitro, cyano and azido, and if R² is cycloalkyl, the groupalso includes C₁-C₆alkyl or C₁-C₆haloalkyl.
 7. A compound according toclaim 6, wherein R² is C₁-C₂₀ alkyl which is substituted orunsubstituted.
 8. A compound according to claim 6, wherein R² is C₁-C₆alkyl, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, n-pentyl and n-hexyl, any of which alkyl speciesbeing optionally substituted with 1, 2 or where valance permits 3substituents independently selected from C₁-C₄alkyl, C₁-C₄ haloalkyl,halo, hydroxy, C₁-C₄alkoxy, C₁-C₄hydroxyalkyl and C₁-C₄alkylcarbonyl. 9.A compound according to claim 1, wherein R² is C₃-C₆ cycloalkylC₁-C₃alkyl in which any alkyl or cycloalkyl is optionally substituted with upto three substituents independently selected from the group consistingof halo, —OR⁵′—C(O)OR⁵, —C(O)R⁵, —OC(O)OR⁵, —NR⁵R⁶, —C(O)NR⁵R⁶,—OC(O)NR⁵R⁶ nitro, cyano, and azido, and in the case of cycloalkyl, alsoC₁-C₆ alkyl or C₁-C₆ haloalkyl.
 10. A compound according to claim 9,wherein R² is selected from cyclopropylmethyl, cyclobutylmethyl,cyclopentylmethyl and especially cyclohexylmethyl, any of whichcycloalkyl species being optionally substituted with 1, 2 or wherevalance permits 3 substituents independently selected from C₁-C₄alkyl,C₁-C₄haloalkyl, halo, hydroxy, C₁-C₄alkoxy, C₁-C₄hydroxyalkyl andC₁-C₄alkylcarbonyl.
 11. A compound according to claim 1 wherein, R³ is abond to the adjacent carbon thereby defining a keto group.
 12. Acompound according to claim 1, wherein R³ is H, including thecorresponding enol with the gamma ketone function on the pyran ring. 13.A compound according to claim 1, wherein R³ is derivatised with aprodrug moiety selected from C(O)R⁵, CR^(a)R^(b)OR⁵, CR^(a)R^(b)OC(O)R⁵,CR^(a)R^(b)OC(O)OR⁵ and C(O)CHR⁷NH₂.
 14. A compound according to claim13, wherein: R³ as C(O)R⁵ has R⁵ as methyl, isopropyl, cyclopropyl ortert-butyl, any of which may be optionally fluorinated; R³ asCR^(a)R^(b)OR⁵, has R^(a) and R^(b) as both H or both methyl and R⁵ ismethyl, isopropyl, cyclopropyl or tert-butyl, any of which may befluorinated; R³ as CR^(a)R^(b)OC(O)R⁵, has R^(a) and R^(b) as both H orboth methyl and R⁵ is methyl, isopropyl, cyclopropyl or tert-butyl, anyof which may be optionally fluorinated; R³ as CR^(a)R^(b)OC(O)OR⁵, hasR^(a) and R^(b) as both H or both methyl and R⁵ is methyl, isopropyl,cyclopropyl or tert-butyl, any of which may be optionally fluorinated;or R³ as C(O)CHR⁷NH₂, especially wherein R⁷ is the side chain of anL-amino acid such as alanine, valine, leucine or isoleucine.
 15. Acompound according to claim 1 wherein R⁴ is H.
 16. A compound accordingto claim 1, wherein R⁴ is derivatised with a prodrug moiety selectedfrom the group consisting of C(O)R⁵, CRaRbOR⁵, CRaRbOC(O)R⁵,CRaRbOC(O)OR⁵ and C(O)CHR⁷NH₂; wherein R^(a) and R^(b) are independentlyselected from H and methyl.
 17. A compound according to claim 16,wherein: R³ as C(O)R⁵ has R⁵ as methyl, isopropyl, cyclopropyl ortert-butyl, any of which may be optionally fluorinated; R³ asCR^(a)R^(b)OR⁵, has R^(a) and R^(b) as both H or both methyl and R⁵ ismethyl, isopropyl, cyclopropyl or tert-butyl, any of which may befluorinated; R³ as CR^(a)R^(b)OC(O)R₅, has R^(a) and R^(b) as both H orboth methyl and R₅ is methyl, isopropyl, cyclopropyl or tert-butyl, anyof which may be optionally fluorinated; R³ as CR^(a)R^(b)OC(O)OR₅, hasR^(a) and R^(b) as both H or both methyl and R₅ is methyl, isopropyl,cyclopropyl or tert-butyl, any of which may be optionally fluorinated;or R³ as C(O)CHR⁷NH₂, especially wherein R⁷ is the side chain of anL-amino acid such as alanine, valine, leucine or isoleucine.
 18. Acompound according to claim 1, wherein in R⁵ and R⁶, the C₁-C₆alkyl orcycloalkyl is substituted with one or more selected from the groupconsisting of halo, hydroxy, C₁-C₄alkoxy, C₁-C₄haloalkyl,hydroxyC₁-C₄alkyl, C₁-C₄alkylcarbonyl, SH, SMe, COOH, COOC₁-C₄alkyl andCONH₂.
 19. A compound according to claim 1, wherein in R⁷, theC₁-C₆alkyl or C₃-C₆cycloalkyl is substituted with one or more from thegroup consisting of halo, hydroxy, phenyl, C₁-C₄alkoxy, C₁-C₄haloalkyl,hydroxyC₁-C₄alkyl, C₁-C₄alkylcarbonyl, SH, SMe, COOH, COOC₁-C₄alkyl andCONH₂.
 20. The compound according to claim 1, wherein said compound is(E)-3-butyryl-6-(non-1-en-1-yl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one ora pharmaceutically acceptable salt, stereoisomer, N-oxide or hydratethereof.
 21. A compound according to claim 1, in combination with apharmaceutically acceptable excipient.
 22. A pharmaceutical compositioncomprising a compound as defined in claim 1 or a pharmaceuticallyacceptable salt, stereoisomer, N-oxide or hydrate thereof, and apharmaceutically acceptable carrier, vehicle or diluent therefor.
 23. Amethod for inhibiting bacterial growth in a subject suffering from abacterial infection, comprising administering a therapeuticallyeffective amount of a compound of Formula I, according to claim 1, or atherapeutically acceptable salt thereof, to the subject.
 24. A methodfor inhibiting bacterial growth in a subject at risk for bacterialcolonisation, comprising administering a therapeutically effectiveamount of a compound of Formula I, according to claim 1, or atherapeutically acceptable salt thereof, to the subject.
 25. The methodaccording to claim 23, wherein said bacterial infection is aGram-positive bacterial infection.
 26. The method according to claim 24,wherein said bacterial colonization is a Gram-positive bacterialcolonization.
 27. A method for inhibiting bacterial growth on skin as apreoperative measure for surgical site infection prevention, comprisingadministering a therapeutically effective amount of a compound ofFormula I according to claim 1, or a therapeutically acceptable saltthereof, to a subject.
 28. A pharmaceutical composition comprising acompound according to claim 1, wherein said composition is in a formsuitable for nasal application against Staphylococcus aureus.
 29. Apharmaceutical composition comprising a compound according to claim 1 incombination with mupirocin.